Pharmaceutical composition comprising particles comprising a complex of a double-stranded polyribonucleotide and a polyalkyleneimine

ABSTRACT

The present invention relates to compositions comprising complexes that are formed by polyinosinic-polycytidylic acid with a polyalkyleneimine, such as polyethyleneimine, that present uniform structural and functional features, as well as to processes for the preparation of said compositions that comply with regulatory requirements. The present invention additionally relates to use of said compositions as medicaments (in particular for treating cancer), alone or in combination with other therapeutic agents and/or in specific medical methods. Moreover, the administration of these compositions is associated to changes in the expression of specific genes, in cell responses, and/or in composition of immune cell populations that can be used as specific biomarkers and/or as additional target for medical treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry under 35 U.S.C. § 371 ofInternational Patent Application PCT/EP2017/079688, filed Nov. 17, 2017,published as International Patent Publication WO 2018/210439 on Nov. 22,2018, which claims the benefit of European Patent Application EP17171617.8, filed on May 17, 2017, European Patent Application EP17382301.4, filed on May 26, 2017, and European Patent Application EP17200469.9, filed on Nov. 7, 2017, the contents of all are herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the field of pharmaceuticalformulations comprising particles formed by polyribonucleotides andpolymers, their production, and their medical use.

BACKGROUND OF THE INVENTION

The use of synthetic analogs of double-stranded RNA (dsRNA) that mimicviral dsRNA has been explored in recent years for specificallyactivating the immune system against tumors with the effect ofinhibiting cancer cell growth and inducing cancer cell apoptosis. Inparticular, double-stranded polyinosinic-polycytidylic acid (known aspoly(I:C) or pIC) has been characterized as a type of dsRNA with variouseffects of therapeutic interest against several types of cancers (suchas melanoma, hepatoma, colon, gastric, and oral carcinoma, cervicalcancer, breast cancer, ovarian cancer, urinary tract tumors, lung andprostate cancer) and their metastasis, in manners that may be dependentor independent from immune system activation, natural killer- and/ordendritic cell-mediated activities, and/or changes of tumor geneexpression and microenvironment (Hafner A et al., 2013).

Unfortunately, these initial preclinical reports are poorly or notconfirmed in clinical studies with naked poly(I:C) molecules, which havedemonstrated their low stability, poor homogeneity, unpredictablepharmacokinetics, and limited anti-tumoral effects due to a variety ofmechanisms, such as poor cellular uptake or degradation by cytosolicRNases (Hafner A et al., 2013). Indeed, in order to achieve an effectivetherapeutic or prophylactic effect, poly(I:C) molecules may need to bere-dissolved immediately prior or shortly before use, may be availablein formulations at low concentrations, and/or must be frequentlyadministered (e.g. every 2 hours).

During the last few years, there has been significant progress informulating poly(I:C) molecules with immunomodulatory and/or therapeuticproperties. Various methods of preparing and formulating poly(I:C)molecules as powder and/or integrated within polymer-basedmicroparticles with or without targeting moieties and additionalchemical linkers have been disclosed (CN103599071; CN102988303;WO2004045491; WO2008057696; WO2013164380; WO2015067632, WO2014057432;WO2014165296; Schaffert D et al., 2011; WO2015173824, Kabilova T et al.,2014; Kithler K et al., 2011; Palchetti S et al., 2013; Saheki A et al.,2011). Poly(I:C) molecules have been formulated with carrier polymersand in formats compatible for nasal administration (WO2013164380),stabilized with polylysine and carboxymethylcellulose (WO2005102278),encapsulated within cationic lipid-coated calcium phosphatenanoparticles, liposomes, or other vesicular structures (Chen L et al.,2013; US2009117306; US2011003883), or together with single stranded RNAand with cationic peptides like protamine (WO2013087083). Alternatively,poly(I:C) molecules have also been immobilized on solid particles andcarriers such as iron oxide nanoparticles, with or without agents thatwould help targeting poly(I:C) molecules to specific cells or tissues(McBain S et al., 2007; Cobaleda-Siles Metal., 2014).

Some publications further disclose various ternary or quaternarycomplexes in the sub-micrometer range that are formed by polymers,poly(I:C) molecules, and/or double stranded DNA, with or without othercomponents and gene-specific (Kurosaki T et al., 2009.; WO2013040552;WO2013063019; Tutin-Moeavin I et al., 2015). However, these approacheshave the objective of providing agents that essentially administer DNAto the cells, while maintaining their viability, and not the selectivekilling of cancer cells.

The pitfalls that are limiting the clinical development of poly(I:C)molecules as a drug and its compliance with demanding regulatoryrequirements could be overcome by producing structurally complexanticancer complexes comprising poly(I:C) molecules together with drugdelivery systems for cancer therapy that are often based on cationicpolymers such as chitosan, polyethyleneimine (PEI), poly-L-lysine,polymethacrylates, imidazole- or cyclodextrin-containing polymers,poly(beta-amino ester)s, and related dendrimers. These polymeric systems(also called as Polyplex) are structurally and functionally distinctfrom lipid-based systems (also called as Lipoplex) and hybrid systems(also called as Lipopolyplex) that are similarly used for the local orsystemic delivering of nucleic acids (Bilensoy E, 2010; Germershaus Oand Nultsch K, 2015). Among Polyplex, PEI is a polyalkyleneimine being acationic polymer of particular interest that can be modified at thelevel of linear/branched structure and size, chemical linkage,degradability, and derivatization (Islam M et al., 2014) and that,differently from lipoplex internalization by cells, is internalized bothby clathrin-mediated and by caveolae mediated endocytosis (Shabani M etal., 2010).

This therapeutic approach involving the preparation and theadministration of poly(I:C) molecules associated to PEI has beenexemplified in the literature by the agent called BO-110 (Pozuelo-RubioM et al., 2014; Tormo D et al., 2009; WO2011003883). This complex, alsoidentified as [pIC]^(PEI), not only engages a dual induction ofautophagy and apoptosis in several cancer cell lines of melanoma and ofother tumor types (such as gliomas or carcinomas) but also has no orlimited effect on the viability of normal cells, such as melanocytes.BO-110 inhibits melanoma growth in animal models for demonstratingantitumoral and antimetastatic activity in vivo, even in severelyimmunocompromised mice. Moreover, a similar [pIC]^(PEI)-based agentstimulates the apoptosis in pancreatic ductal adenocarcinoma cellswithout affecting normal pancreatic epithelial cells and in vivoadministration of [pIC]^(PEI) inhibited tumor growth in tumor animalmodels (Bhoopathi Petal., 2014). A further effect of BO-110administration is characterized in a model of endometriosis, whereinsuch agent reduces angiogenesis and cellular proliferation and increasesapoptosis (Garcia-Pascual C and Gomez R, 2013). Thus, BO-110 and similar[pIC]^(PEI) agents that comprise double-stranded polyribonucleotidesrepresent a novel anticancer strategy with a broad spectrum of action,due to the combined activation of autophagy and apoptosis, autonomouslyand selectively in tumor cells, while maintaining the viability ofnormal cells of different lineages. However, BO-110, as for otherdouble-stranded polyribonucleotide-based agents that have demonstratedefficacy in various pre-clinical models when associated with carriers,still needs to be provided in formulations that are stable in differentstorage conditions, uniformly manufactured and sized, and more readilyadapted to medical uses (in particular those directed to cancer) withrespect to most effective combinations, regimens, dosages, and clinicalfollow-up when using other drugs and therapies.

Indeed, prior art does not provide appropriate teaching for solvingissues related to the most effective combination of structural andbiophysical criteria that allow the production of poly(I:C)-containingcompositions for treatment of cancer. Regulatory agencies also requirebeing strictly compliant to the specifications on reproducibility,storage, and uniformity of the size and concentration ofpoly(I:C)-containing particles that are included within compositions foruse in humans. The general features of formulations of double-strandedpolyribonucleotide-based (such as poly(I:C) molecules) and relatedagents, compositions (collectively identified as BO-11X products), andrelated processes providing double-stranded polyribonucleotidemolecules, at higher, and well-controlled, concentrations were describedin the PCT application PCT/EP2016/078078. However, the pre-clinical andclinical characterization of BO-11X products are still needed to allowits effective medical use as a drug (in particular against cancer) inconnection to specific indications, ongoing treatments, and/or regimens,while improving means for clinical evaluation and use together withpatient compliance, and reducing the frequency of dosing double-strandedpolyribonucleotide molecules with well-defined safety margin andtherapeutic effects.

SUMMARY OF THE INVENTION

The present invention relates to a composition comprising particleswherein: (i) each of said particles comprises a complex of at least onedouble-stranded polyribonucleotide, or a salt or solvate thereof, and atleast one polyalkyleneimine, or a salt and/or solvate thereof; (ii) atleast 95%, or at least 90%, of said particles has a diameter of lessthan or equal to 600 nm, preferably, less than or equal to 300 nm (forexample, between 140 and 250 nm); and (iii)said particles have az-average diameter of less than or equal to 200 nm, preferably less thanor equal to 150 nm, in particular, as measured according to ISO22412:2017.

In a preferred embodiment, the present invention relates to an aqueouscomposition comprising particles wherein: (i) each of said particlescomprises a complex of at least one double-stranded polyribonucleotide,or a salt or solvate thereof, and at least one linear polyalkyleneimine,or a salt and/or solvate thereof, wherein said double-strandedpolyribonucleotide is polyinosinic-polycytidylic acid [poly(I:C)] andthe average molecular weight of said linear polyalkyleneimine is between17 and 23 kDa; (ii) at least 90% of said particles has a mono-modaldiameter distribution below 300 nm; (iii) said particles have az-average diameter of less than or equal to 200 nm, as measuredaccording to ISO 22412:2017; and (iv) said composition has a zetapotential equal or superior to 30 mV, preferably between 35 and 50 mV,according to ISO 13099-2:2012.

The present invention also relates to an aqueous composition comprisingparticles as disclosed herein wherein: (i) each of said particles isformed by making a complex of at least one double-strandedpolyribonucleotide, or a salt or solvate thereof, and at least onelinear polyalkyleneimine, or a salt and/or solvate thereof, wherein saiddouble-stranded polyribonucleotide is polyinosinic-polycytidylic acid[poly(I:C)] and the average molecular weight of said linearpolyalkyleneimine is between 17 and 23 kDa; (ii) at least 90% of saidparticles has a mono-modal diameter below 300 nm; (iii) said particleshave a z-average diameter of less than or equal to 200 nm, as measuredaccording to ISO 22412:2017; and (iv) said composition has a zetapotential equal or superior to 30 mV, preferably between 35 and 50 mV,according to ISO 13099-2:2012; wherein said particles are formed at theratio of the number of moles of nitrogen of said polyalkyleneimine tothe number of moles of phosphorus of said double-strandedpolyribonucleotide in said composition being equal to or greater than2.5.

The present invention also relates to a composition obtainable bylyophilisation of the aqueous composition as disclosed herein.

These compositions can be further defined on the basis of the featuresassociated to the following criteria: a pH comprised between 2 and 4 anda concentration of one double-stranded polyribonucleotide equal orsuperior to 0.5 mg per mL of the total volume of said composition.

These compositions can be further defined on the basis of amount andsize of the double-stranded polyribonucleotides, in particular: (i) atleast 40% of the double-stranded polyribonucleotides comprised in saidparticles have at least 850 base pairs, and at least 50% of thedouble-stranded polyribonucleotides comprised in said particles havebetween 400 and 5000 base pairs; and/or (ii) between 5% and 60% ofdouble-stranded polyribonucleotides having less than 400 base pairs,between 15% and 30% of double-stranded polyribonucleotides havingbetween 400 and 850 base, between 10% and 70% of double-strandedpolyribonucleotides having between 850 and 5000 base pairs, and between0% and 10% of double-stranded polyribonucleotides having more than 5000base pairs.

These compositions have been successfully optimized for pharmaceuticalmanufacturing and clinical use, in particular by identifying the optimaland more reproducible combinations of components, physical and/orchemical criteria, and related numerical ranges (applicable to eitherthe particles or the compositions) for the desired uses and methods.Thus, a further preferred composition is an aqueous compositioncomprising particles wherein: (i) each of said particles comprises acomplex of (a) polyinosinic-polycytidylic acid [poly(I:C)], or a salt orsolvate thereof, wherein at least 40% of poly(I:C) molecules comprisedin said particles have at least 850 base pairs, and at least 50% ofpoly(I:C) molecules comprised in said particles have between 400 and5000 base pairs, and (b) a water-soluble, linear homo-polyalkyleneimineor hetero-polyalkyleneimine, or a salt and/or solvate thereof, whereinthe average molecular weight of said linear polyalkyleneimine is between17 and 23 kDa (ii) at least 90% of said particles has a mono-modaldiameter distribution below 300 nm (and preferably below 250 nm); (iii)said particles have a z-average diameter of between 60 nm and 130 nmdiameter (and more preferably of 80+/−20 nm), as measured according toISO 22412:2017, with polydispersity index of said particle diameterwhich is inferior to 1.5; (iv) said composition contains poly(I:C) at aconcentration of at least 0.5 mg/mL; (v) said composition has: a pH ofbetween 2 and 4 and osmolality of between 200 and 600 mOsm/kg; and (vi)said composition has a zeta potential between 35 mV and 50 mV, accordingto ISO 13099-2:2012.

These ranges identified in aqueous composition and particles definedabove can be further defined and/or limited to: (i) particles comprisingat least 10% of poly(I:C) molecules have less than 400 base pairs, atleast 40% of poly(I:C) molecules having at least 850 base pairs, atleast 70% of poly(I:C) molecules having between 400 and 5000 base pairs,and between 20% and 45% of poly(I:C) molecules having between 400 and850 base pairs; (ii) particles comprising between 5% and 60% ofpoly(I:C) molecules having less than 400 base pairs, between 15% and 30%of poly(I:C) molecules having between 400 and 850 base, between 10% and70% of poly(I:C) molecules having between 850 and 5000 base pairs, andbetween 0% and 10% of poly(I:C) molecules having more than 5000 basepairs. (iii) said particles have a median diameter (D50%) of between 75nm and 150 nm (and preferably at least have a median diameter of 85+/−20nm); (v) the water-soluble, linear homo-polyalkyleneimine orhetero-polyalkyleneimine being a linear polyethyleneimine; (vi) thepolydispersity index of said particle diameter is comprised between 0.2and 0.3; (vii) the composition having a pH 3.0+/−0.2 and an osmolalityof between 300 and 310 mOsm/kg.

In addition, the present invention relates to a composition, i.e. BO-11Xformulations or compositions as disclosed herein, for use as amedicament, alone in combination with other therapeutic agents. Thesecompositions may further comprise at least one pharmaceuticallyacceptable carrier, organic solvent, excipient and/or adjuvant, eitherincluded in the particles themselves or added in the aqueouscomposition, for example glucose added in a concentration of between 1and 10% (weight/volume). Moreover, the composition may further compriseat least one compound, and in particular a therapeutic compound,selected from an organic compound, an inorganic compound, a nucleic acid(for example, non-coding RNA or RNA coding for proteins), an aptamer, apeptide or a protein, either included in the particles themselves oradded in the aqueous composition.

Moreover, this composition can be administered for medical uses usingregimens that combine different routes of administration (e.g. one ormore intra-tumoral injections that are followed by one or moresub-cutaneous or intra-muscular injections over a period of 1 or moreweeks) and/or the ex vivo exposure of human cells to the composition,prior to re-administering the cells to the patient. In this latter case,such regimen involves the induction of specific activities within thecells of the patient that are exposed to the composition, for instance,the induction of interferon production by said cells in vitro.Otherwise, in vitro and/or ex vivo studies with respect to immuneresponse to such composition show that BO-11X compositions trigger otherbiological mechanisms (interferon-independent and/or targeting immunecells, for example) that can be exploited for promoting therapeuticallyrelevant events such as tumor cell death, enhanced local and/or systemicT cell immune response either directly (within injected tumors) or indistant tumors, and other mechanisms that may be useful for treatingcancers that are recurrent, unresponsive or refractory to othertherapies.

The dose of the composition, in particular with respect to the contentof double-stranded polyribonucleotides, can be adapted consequently toeach type of administration, regimen (e.g. highest for intra-tumoralinjection, lower for sub-cutaneous or intra-muscular injection, and evenlower for treating cells ex vivo), and/or other drugs (when administeredin combination therapies).

The present invention also relates to a composition, as disclosedherein, for use in treatment or prevention of a cell growth disordercharacterized by abnormal growth of human or animal cells, preferablycancer, and most preferably solid cancers or lymphomas. Furthermore,this composition can be administered for supporting vaccines, cytokines,antigens, antibodies, chemical compounds, and other compounds havingimmunomodulatory activities for treating or preventing cancers (solid ornot) or infection, for instance as adjuvant and/or for rescuing patientspoorly responding or resistant to a drug, including agents for cancerimmunotherapy, for altering cell metabolism and/or functions(preferably, in immune and/or cancer cells), for modulating DNAexpression, replication and/or repair (including drugs that targetepigenetic mechanisms), or for standard-of-care therapies (such aschemo- or radiotherapy, or vaccine-based therapies involving cancer orviral antigens).

Other objects of the present invention are related to the methodsrelated to the composition, as disclosed herein, for evaluating theefficacy, the most appropriate regimen, the most appropriate therapeuticcombination with another anti-cancer drug or standard-of-care protocol,and/or subjects presenting the best response to the treatment with aBO-11X treatment.

These methods involve measuring the up- and down regulation in theexpression of panels of genes in selected cell types (such as immunecells and cancer cells) following to the exposure to a BO-11Xcomposition and consequently applying appropriate means for improvingtherapeutic efficacy (e.g. for stratifying or selecting patients forfurther treatments, administering or not drugs targeting specificbiological targets, and/or reducing or increasing the dosage of BO-11Xand/or other compositions).

Furthermore, the present invention relates to a process of manufacturinga composition, i.e. BO-11X formulations or compositions as disclosedherein, which comprises: (i) preparing an aqueous solution of at leastone double-stranded polyribonucleotide, or a salt or solvate thereof,and an aqueous solution of at least one polyalkyleneimine, or a salt orsolvate thereof, wherein either or both solutions optionally furthercomprise a pharmaceutically acceptable carrier, organic solvent,excipient and/or adjuvant; (ii) independently filtering each solutionthrough a filter having a pore diameter of less than or equal to 500 nmto form sterilized solutions; (iii) mixing the resulting sterilizedsolutions in a mixing chamber to form an aqueous composition by additionof one of said solutions into the other solution in said mixing chamber,optionally by injection, at a rate of greater than or equal to 1 mL/min,or by simultaneous addition of each of said solutions into said mixingchamber, optionally by injection, at a rate of greater than or equal to1 mL/min; and optionally (iv) filtering the resulting aqueouscomposition through a filter having a pore diameter of less than orequal to 600 nm to form a filtrate, or centrifuging the resultingaqueous composition at greater than or equal to 22480 m/s² to form asupernatant.

The present invention also relates to the aqueous composition obtainableby said process (including the compositions that can be defined as lightsuspensions), as well as the compositions that can be optionallyobtained by lyophilising the resulting aqueous composition, filtrate orsupernatant and then provided and/or used separately (or in kits) withother medical compounds or devices (such as buffers, diluents,catheters, needles, filters, or devices adapted for intratumoraladministration).

Preferably, the present invention also relates to a compositioncomprising particles wherein: (i) each of said particles comprises acomplex of at least one double-stranded polyribonucleotide, or a salt orsolvate thereof, and at least one polyalkyleneimine, or a salt and/orsolvate thereof, wherein (a) said double-stranded polyribonucleotide ispolyinosinic-polycytidylic acid [poly(I:C)] or polyadenylic-polyuridylicacid [poly(A:U)], wherein at least 10% of double-strandedpolyribonucleotides have less than 400 base pairs, at least 40% of saiddouble-stranded polyribonucleotides have at least 850 base pairs, atleast 70% of said double-stranded polyribonucleotides have between 400and 5000 base pairs, and between 20% and 45% of said double-strandedpolyribonucleotides have between 400 and 850 base pairs; and (b) saidpolyalkyleneimine comprises at least 95% polyethyleneimines, wherein theweight average molecular weight of said polyalkyleneimine is between 17and 23 kDa and the polydispersity index is <1.5, and wherein the ratioof the number of moles of nitrogen of said polyalkyleneimine to thenumber of moles of phosphorus of said double-stranded polyribonucleotidein said composition is between 2.5 and 5.5; (ii) at least 99% of saidparticles has a diameter of less than or equal to 600 nm; and (iii) saidparticles have a z-average diameter of between 30 nm and 150 nm.

More preferably, the present invention also relates to an aqueouscomposition which comprises particles wherein: (i) each of saidparticles comprises a complex of at least one double-strandedpolyribonucleotide, or a salt or solvate thereof, and at least onepolyalkyleneimine, or a salt and/or solvate thereof, wherein (a) saiddouble-stranded polyribonucleotide is polyinosinic-polycytidylic acid[poly(I:C)], wherein at least 10% of double-stranded polyribonucleotideshave less than 400 base pairs, at least 40% of said poly(I:C) has atleast 850 base pairs, at least 70% of said poly(I:C) has between 400 and5000 base pairs, and between 20% and 45% of said poly(I:C) has between400 and 850 base pairs; and (b) said polyalkyleneimine ispolyethyleneimine (PEI), wherein the weight average molecular weight ofsaid PEI is between 17.5 and 22.6 kDa and the polydispersity index is<1.5, and wherein the ratio of the number of moles of nitrogen of saidpolyalkyleneimine to the number of moles of phosphorus of saiddouble-stranded polyribonucleotide in said composition is between 2.5and 4.5; (ii) at least 99% of said particles has a diameter of less thanor equal to 500 nm; (iii) said particles have a z-average diameter ofbetween 60 nm and 130 nm; and (iv) said particles have a median diameter(D50%) of between 75 nm and 150 nm.

Any of the compositions as defined above can be further defined withrespect to features of the manufacturing process, including compositionsobtainable by lyophilisation of the aqueous composition defined above.For example, the particles are formed at the ratio of the number ofmoles of nitrogen of said water-soluble, linear homo-polyalkyleneimineor hetero-polyalkyleneimine to the number of moles of phosphorus ofpoly(I:C) molecules between 2.5 and 4.5. Preferably, the particles areformed by injecting separately the solution containing said poly(I:C)molecules and the solution containing said water-soluble, linearhomo-polyalkyleneimine or hetero-polyalkyleneimine in a mixing chamber.The aqueous compositions can be formed by adding glucose in aconcentration of between 1 and 10% (weight/total volume of saidcomposition), preferably by adding glucose to the solution containingpoly(I:C). Moreover, wherein the solution containing said poly(I:C)molecules and the solution containing said water-soluble, linearhomo-polyalkyleneimine or hetero-polyalkyleneimine are injectedseparately, wherein the flow speed for injecting either solution isbetween 1 mL/min and 50 mL/min, and/or mixed at a speed between 50 rpmand 600 rpm.

The more preferred ranges that are identified above within (i)-(vii)above with respect to the sizes of poly(I:C) molecules contained in theparticles, the mono-modal diameter distribution of particles the linearpolyalkyleneimine being polyethyleneimine, the polydispersity index ofparticle diameter, the osmolality of composition, and/or pH ofcomposition (as well the presence one pharmaceutically acceptablecarrier, organic solvent, excipient and/or adjuvant, or the presence ofat least one compound, and in particular a therapeutic compound) alsoapply to the aqueous compositions which comprises particles that areformed as indicated above.

Further embodiments related to the preparation of such compositions inform of BO-11X formulations, their features, their analysis, and theiruses (alone or in combination with other agents, as well as in specificregimens) are provided in the Detailed Description and in the Examplesbelow, in particular for obtaining improved or optimized therapeuticresponses for specific indications and/or in combination with otherdrugs and therapies (such as chemotherapy, radiotherapy, agents thattarget immune checkpoint molecules, T cell-mediated responses, DNArepair and/or replication, or inhibitors of kinases and other enzymesthat modulate activities in immune cells and/or cancer cells, includingmetabolic activities). BO-11X formulations can be used in methods fortreating a cell growth disorder characterized by abnormal growth ofhuman or animal cells (and in particular cancer) in combination with asecond therapeutic agent selected from anti-CTLA4, anti-PD1, anti-PDL1,CAR-T cells, cancer antigen vaccines, or agents that target regulatory Tcells, metabolic enzymes, DNA repair and/or replication, or a proteinexpressed by any of the genes of Table I (see Example 4). Moreparticularly, BO-11X formulations can used for obtaining a synergistictherapeutic effect when administered with this second therapeutic agent,including the possibility of reducing the regular dosage and/orfrequency of administration of said second therapeutic agent (thuspotentially reducing medical interventions, resistance to drugs, and/orpatient's discomfort). Moreover, BO-11X formulations when administeredwith this second therapeutic agent, may allow treating patients that areresistant, insensitive, or poorly (or not) responding to said secondtherapeutic agent, overcoming any specific tumor resistance or escapemechanism (including mutations that alter specific genes, pathways,and/or response to drugs or endogenous compounds such as cytokines).Thus, BO-11X formulations can be used in the form of a drug-rescuing ordrug-sensitizing combination treatment, in particular for treatingcancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: GMP manufacturing and structural analysis of BO-11X aqueouscompositions, as exemplified by BO-112. (A) Flowchart summarizing mainsteps for manufacturing BO-112 compounds as a GMP-compliant,pharmaceutical preparation comprising poly(I:C) molecules, commercialJetPEI preparations, and glucose. This approach can be applied also byusing other types of polyribonucleotides that form double strandedmolecules that are formed by Poly (A), poly(U), poly (C), and/or Poly(G) according to their respective pairing properties. Otherwise, thisapproach may involve adding another compound (being an immune-relatedadjuvant or therapeutic compound, such as CD40L, IL12, viral or tumorantigens, miRNA, mRNA, or other (non-coding) RNA-/nucleic acid-baseddrugs, or an anti-cancer drug such as inhibitors of kinases or otherenzymes affecting cell replication, metabolism, or biological functions)together with (or as an alternative to) glucose or other excipient inStage 1. (B) Size distribution of BO-112 complexes is compared to threepoly(I:C)-containing commercial products (Poly-ICLC, LyoVec-HMW, andLyoVec-LMW); particle size is defined considering particle diameter innanometers (d.nm) by Dynamic Light Scattering (zeta sizer nano ZStechnology). These commercial preparations also present substantialchanges in the size distribution following storage at −20° C., thatBO-112 preparations do not present.

FIG. 2: Effect of different poly(I:C) formulations on cell viability indistinct cancer cell models. (A) BO-112 is compared to untreated cellsand to cells treated with Poly-ICLC, LyoVec-LMW, or LyoVec-HMW, eachformulation being tested at the indicated concentrations that weredetermined according to complex weight but with a similar content ofpoly(I:C) molecules in either a melanoma (SK-MEL-103) or pancreaticcancer (PANC 02.03) cell model. The cell viability data were generatedusing crystal violet assay method after 24 hours. This in vitrocytotoxicity assay was performed in other mouse (4T1 breast cancer, MC38colon cancer, and B16-F10-OVA) melanoma cell lines) and human (PAN02.03pancreatic cancer cell line) models by incubating the cells with0.25-1.0 μg/mL of BO-112 for 24 or 48 hours, confirming the directcytotoxic effect on such cell lines. (B) The effect of differentpoly(I:C) formulations on signaling molecules of therapeutic interestsuch as Interferon-beta (IFN-beta) expression was evaluated by RT-qPCRmethod in SK-MEL-103 cells that were exposed to BO-112, Poly-ICLC (oruntreated, NT) for 8, 16 and 24 hours. BO-112 and Poly-ICLC formulationswere used at a concentration providing a similar content of poly(I:C)molecules.

FIG. 3: Effect of linear PEI alone, poly(I:C), and two differentPEI/poly(I:C) formulations (BO-110 and BO-112) on the viability ofnormal, primary melanocytes (preparations #1 and #2) and four cancercell lines, demonstrating the specific cytotoxicity of suchPEI/poly(I:C) formulations against cancer cell lines. Poly(I:C) contentused in preparation of the Poly(I:C) only, BO-110, and BO-112 treatmentsadministered is identical (1 μg/mL per 40 hours of treatment).

FIG. 4: Effect of BO-112 different poly(I:C) formulations onpatient-derived melanoma cells (SK-Mel 103 cell line). BO-112 iscompared ex vivo to untreated cells (NT) and to cells treated withPoly-ICLC, LyoVec-LMW, or LyoVec-HMW, each formulation being tested atthe concentrations that were determined according to complex weight butwith a similar content of poly(I:C) molecules. (A) The cell viabilitydata were generated using crystal violet assay method after a 48 hourtreatment with each poly(I:C) formulation at 0.35 μg/ml concentration.(B) The expression of Interferon-alpha/beta (IFNa/b, in Arbitrary Units)was evaluated at 16 and 24 hours after treatment with each poly(I:C)formulation at 0.5 μg/ml concentration.

FIG. 5: Effect of BO-112, lipopolysaccaharides (LPS), and poly(I:C)preparation for producing BO-112 on different type of cell death and theexpression of related markers. B16-F10-OVA (cells 10⁵ cells/well) werecultured alone (control) or with LPS, BO-112 or poly(I:C) molecules only(having the same size distribution of those incorporated in BO-112formulations), at the indicated concentrations for 48 hours, measuringby flow cytometry the percentage of cells still viable or presentingcombinations of markers related to early/late apoptosis, or necrosis(annexin V and 7ADD staining; A), or expressing specific markers ofimmunogenic cell death such as MHC-I, CD95 or Calreticulin, (B).

FIG. 6: Effect of BO-112 on activation of the enzyme Poly ADP ribosepolymerase (PARP). 4T1 breast or MC38 colon cancer cells (3×10⁶) werecultured with BO-112 (0.5 μg/mL). Activation of PARP, an enzyme which isinvolved in DNA repair, genomic stability, and programmed cell death wasevaluated at 0, 16 h and 24 h after BO-112 stimulation by WesternBlotting analysis. It was analyzed the presence in the cell lysates ofthe complete 116 kDa PARP protein and the 85 kDa apoptosis-inducedcleavage product; tubulin was included as control for loading equalprotein amounts. Results show that BO-112 formulation promotes theactivation of PARP enzyme in both MC38 and 4T1 cancer cells, providingevidence for use of BO-112 composition in combination with PARPinhibitor in patients where this latter type of drugs is poorly or noteffective and may require being sensitized to (see also FIGS. 14B and15B).

FIG. 7: Effect of BO-112 administration in an animal model for humanmelanoma. (A) Timeline showing the schedule of treatment. All mice wereinjected sub-cutaneously with B16-F10 murine melanoma cells at day 0.After randomizing mice into five groups presenting tumors of similaraverage size (80-100 mm³) on day 7, animals were treated with BO-112formulation (double circles) by injection straight into the tumor tissue(i.t. treatment) at 3 different concentrations in groups 2, 3, and 4(G2, G3, G4) or, in the two remaining groups (G1 and G5), with vehicleonly. On the following treatment days (10, 14, 17, 21 and 24) all groupsbut G1, received intraperitoneal (i.p.) administration of an anti-PD-L1murine antibody (150 μg/dose) in addition to the intratumoraladministration of BO-112 formulation at the same concentration of day 7.Survival was monitored daily, and mice were scored as dead upon findingthem deceased, or when the tumor volume reached the maximum allowedsize. Monitoring continued after last treatment until day 45, when thelast mouse died and the experiment was terminated. (B) Survival curvecomparing the control groups G1 and G5 with the three groups in whichBO-112 formulation was administered at the indicated threeconcentrations. When comparing the groups, there was a statisticaldifference (p<0.0001, Log-rank Mantel-Cox test) between the controlgroups and the test groups, with G4 showing the strongest increase ofsurvival relative to vehicle or anti-PD-L1 alone. (C) Therapeutic effectof BO-112 as single agent in a further B16-F10 model for human melanoma;B16-F10 melanoma cells (5×10⁵ cells) are injected sub-cutaneously andBO-112 is administered intratumorally as described in A (8-9 mice/group;control group, vehicle only; treatment group, BO-112; BO-112 dose/mouse:2.5 mg/Kg in 100 μl). (D) The size of tumors was measured by calliperweekly and volume calculated (length×width²/2) for each animal. Spiderplots show individual tumor growth curves for control (vehicle) andBO-112 treated mice and indicate that BO-112 formulation reduces tumorgrowth compared to vehicle.

FIG. 8: Abscopal effect of BO-112 administration in an animal model forhuman melanoma. (A) Mice in which two cancers are induced bytransferring melanoma B16-F10-OVA are treated in only one one tumor massby intratumoral administration (for BO-112) and/or systemically byintraperitoneal administration (for anti-PD-L1), using a regimen anddosage similar to those of the experiment shown in FIG. 7 (n=10mice/group). (B) The growth of tumor volume was measured in both thetreated and untreated, distal tumor masses in the four groups: control,single treatment, or combined treatment. Tumors were measured bycalliper weekly and volume calculated (length×width²/2) for each animalin a group.

FIG. 9: Effect on BO-112 administration in an animal model for humanbreast cancer. (A) Model and treatment scheme using 4T1 breast carcinomacells (5×10⁵ cells) that were injected sub-cutaneously (s.c.) in theright flank of 8- to 10-week-old female BALB/c mice (6-7 mice/group;control group, vehicle only; treatment group, BO-112). Treatmentsstarted when tumor volume was 80-100 mm³ (on day 9). BO-112administration and vehicle was performed intratumorally via a singledirect injection into the tumor mass of the right flank (dose/mouse: 2.5mg/Kg in 100 μI) at the indicated days. Tumors were measured by calliperweekly and volume calculated (length×width²/2) for each animal in agroup, obtaining data that are shown separately for each of such animals(the number of animals for each group is indicated (B), or inconsolidated for each group (C), observing a statistically relevantdecrease of tumor volume in the BO-112 treated group.

FIG. 10: Effect on BO-112 administration in an animal model for humancolorectal cancer. (A) In a first model and treatment scheme, MC38 coloncarcinoma cells (5×10⁵ cells) were injected s.c in the right flank of 8-to 10-week-old female C57BL/6 mice (7-8 mice/group; G1 control group,vehicle only; G2 treatment group). Treatments started when tumor volumewas 80-100 mm³ (on day 16). BO-112 administration and vehicle wasperformed intratumorally via a single direct injection into the tumormass of the RIGHT flank (dose/mouse: 2.5 mg/Kg in 100 μI) at theindicated days. (B) Mouse survival in this first model was evaluatedusing Kaplan-Meier analysis. Tumors were also measured by calliperweekly and volume calculated (length×width²/2) for each animal in agroup obtaining data that are shown separately for each of such animals(C) or in consolidated for each group (D), observing a statisticallyrelevant decrease of tumor volume in the BO-112 treated group.

FIG. 11: Effect on BO-112 administration in a second model for humancolorectal cancer using a different treatment scheme. (A) MC38 coloncarcinoma cells (1×10⁶ cells) were injected s.c in the right flank of 8-to 10-week-old female C57BL/6 mice (4-11 mice/group; G1 control group,vehicle only; G2 treatment group). Treatments started when tumor volumewas 80-100 mm³ (on day 11). BO-112 administration and vehicle wasperformed intratumorally via a single direct injection into the tumormass of the right flank (dose/mouse: 2.5 mg/Kg in 100 μl) at theindicated day. In those remaining mice that were tumor-free after BO-112treatment, a re-challenge was done on day 60 using MC38 cells (2.5×10⁵cells) that were injected s.c in the left flank, following tumor growthand mouse survival. (B) Mouse survival in this second model was alsoevaluated using Kaplan-Meier analysis, with the four animals that werere-challenged at day 60 and still alive at day 80, suggesting thatBO-112 can both trigger apoptosis of cancer cells and drive immunememory against them.

FIG. 12: BO-112 therapeutic effect in models for adaptive immuneresponse activation in human cancers. (A) The MC38-based model for humancolon carcinoma is established as previously described but integratingtwo further groups in which an antibody for depleting either CD4positiveor CD8positive T cells is also administered (treatment schedule: B0112:d8, d11, d15, d18, d22, d25; anti-CD4 or anti-CD8: d7, d8, d11, d15,d22, d29,). (B). Analysis of the tumor growth curves of MC38tumor-bearing mice treated with BO-112, showing that only the depletionof CD8positive T cells diminishes the therapeutic properties of BO-112formulation (tumor volume is shown in parallel for the different groups,N for the number of mice per group and with a dotted line indicating theend of BO-112 treatment at d25). (C) Effect of BO-112 therapy on T cellrecruitment and activation into the tumor, and on the priming of antigenspecific CD8+ T cells. Frequency of different cell markers for T cellsubsets (CD8 positive or CD4 positive) ad T cell activation (CD137,IFN-γ and PD-1) is measured in B16-F10-OVA tumors (and in draining lymphnodes for tetramer analysis) from mice treated with vehicle or BO-112formulation 24 hours after the second BO-112 administration by flowcytometry. The graphs show that BO-112 therapy significantly increasesCD8+ T cells and promotes CD4+and CD8+ T cell activation in the tumorinfiltrate in this model. (D) Frequency of different cell markers for Tcell subsets (CD8 positive or CD4 positive) for T cell priming (OVA andTrp2 tetramers), showing that BO-112 also enhances significantly thefrequency of Antigen-specific CD8+ T cells in the tumor (against OVA andendogenous Trp2 Antigens) and in the draining lymph node (particularlyfor Trp2 Antigen) with significant. All data (even originate fromdifferent experiments) consistently indicate that BO-112 formulationactivates the anti-tumor adaptive immune response with the potential toinduce systemic immunity.

FIG. 13: BO-112 therapeutic effect in models for evaluating dependencyfrom interferons expression in human cancers. (A) The in vitro tumorcell cytotoxicity independent of IFNs is evaluated in terms of cellviability at 24, 48 and 72 h by MTS assay. Briefly, cells from each cellline: B16, B16-IFN α/β (do not respond to IFNγ; from InvivoGen) andB16-IFNγ (do not respond to IFNα/β; from InvivoGen) were cultured (5×10³cells/well, 96 flat-well plates): alone or with BO-112 at 0.5 μg/ml for24, 48, and 72 hours alone or alternatively with BO-112 (0.5 μg/ml), orwith 2′3′cGAMP STING agonist (7 μg/ml) for 24 hours (showing BO-112superiority in efficacy to a reference STING agonist). Two independentassays were performed, with at least 8 wells per condition. The In vivotherapeutic effect of BO-112 on IFNs resistant B16-based model for humanmelanoma were tested in the above described B16-IFN α/β (B) and B16-IFNγ(C) cancer cells were injected s.c in the RIGHT flank (5×10⁵ cells) of8- to 10-week-old female C57BL/6 mice (7-10 mice/group). Treatmentsstarted when tumor volume was 80-100 mm³ (on day 8). BO-112administration and vehicle was performed intratumorally via a singledirect injection into the tumor mass of the RIGHT flank (BO-112dose/mouse: 2.5 mg/Kg in 100 μl; 2 doses/week, 3 weeks). Tumors weremeasured by caliper weekly until sacrificed and volume calculated.Graphs show that BO-112 reduces tumor volume (individual spider plots)and improves significantly survival (Kaplan-Meier curves) in BO-112treated mice compared to vehicle, indicating that BO-112 formulation hasa potent therapeutic effect in IFNs-resistant cancer cells.

FIG. 14: Alternative means and regimens for BO-11X administration. (A)the administration of a BO-11X formulation such as BO-112 may start with1-4 doses of intra-tumoral injections (BO-11XI.T.) to be made everyweek, every two weeks, or every three weeks (0.4-2.4 mg/dose; or with alarger interval of up to every 6 weeks, as it can be done in respondingor stable patients during clinical studies). The initial doses can befollowed by a number of sub-cutaneous or intramuscular injections ofBO-11X (BO-11XSQ/I.M.; 0.2-2 mg dose). As for other intra-tumoraltreatments, this regimen may involve the injection of same lesion (ifstill present) or other lesion if original lesion is no longer presentor if the initial intratumoral injections have not reduced such lesions(through systemic immune activation or other response) or otherevidences of tumor burden, stage, persistence, metastasis, and/orrecurrence. (B) Alternatively, BO-112 administration can be pursued byintratumoral injections that are progressively spaced (every week, everytwo, four, six or more weeks) during 20 or weeks, alternating I.T.administration in the same (black diamonds) and other cancer lesions(grey diamonds), with or without administering in parallel treatmentssuch as chemotherapy, radiotherapy, small molecule, checkpointinhibitor, antibody (triangles, administered once every 2 weeks or onceevery 3 weeks as examples) in other regimens and/or administrations thatare started before or after the BO-11X treatment. In the former case,the initial BO-11X monotherapy regimen A can be combined (for example,with pembrolizumab or ipilimumab) or BO-11X monotherapy regimen B can becombined (for example, with nivolumab) for rescuing or supporting thetreatment of this second therapeutic agent which otherwise would bepoorly (or not) effective. In the latter case, either BO-11X monotherapyregimen A or initial BO-11X monotherapy regimen B (each of two regimenscombined with the antibodies previously cited) allows sensitizing thetumor (or the patient) to the additional therapeutic agent (inparticular, an immunotherapy). (C) As an adjuvant to be used ex vivo oncells obtained from patients (such as a vaccine adjuvant for ex vivomaturation of dendritic cells, DCs), the treatment may start byobtaining a blood sample from a patient for isolating relevant celltypes by cytapheresis, a procedure for separating cells by positiveselection and cell sorting such a subset of Dendritic Cells (DCs), forexample monocyte-derived dendritic cells (Mo-DCs), BDCA3-positive andother subsets of dendritic cells that are major producers of Type IInterferon in response to stimuli such as poly(I-C) molecules, orCD34+-derived dendritic cells. These cells can be then expanded and/ormatured ex vivo for a given number of days, during which cells areexposed to BO-112 formulations for variable number of hours (e.g. 1hour, 3 hrs, 8 hrs, 24 hrs or more), and then used for antigen loadingto complete the preparation of a DCs-based vaccination. Such anactivation of DCs can be also followed and completed (or directlyachieved) through intra-muscular or sub-cutaneous injection of BO-112,the induction of immunogenic cell death in tumors by intra-tumoralinjection of BO-112, and/or by in vivo delivery of tumor antigens.

FIG. 15: Alternative means for evaluating and using BO-11X in patients.(A) Flowchart summarizing medically relevant readouts to evaluateadministration of a BO-11X formulation such as BO-112. Follow-up ofmedical conditions can be performed by means of, for example, physicalexamination and imaging techniques for identifying and measuring tumorburden, and/or monitoring cancer-specific biomarkers in blood toevaluate response to treatment, or recurrence. A series of sub-criteriacan be associated to each of the three main evaluation criteria (i.e.boxes in the bottom of the flowchart), involving the evaluation ofspecific parameters in tumor tissue obtained via biopsy and/or in bloodsamples, including the modification in the expression of and/or responseto cytokines (such as interferons or interleukins, individually or inspecific combinations, see Example 4) or circulating/infiltrating immunecells. (B) Flowchart summarizing therapeutic opportunities that areavailable following (but possibly started even before in concurrentlywith) the administration of a BO-11X formulation such as BO-112,involving the readouts as defined in the previous flowchart andadditional readouts (such as those coming from the analysis of geneexpression, protein expression, mutational burden status, or T cellclonality in specific cells or tissues before and after BO-112administration). This analysis provides BO-112-induced molecularsignatures that may indicate continuation with the same or a differentregimen of BO-11X treatment or a different type of medical follow-up,including the possibility to use treatments (e.g., radiotherapy,chemotherapy, anti-PD-1 or other immunomodulating therapy, cancervaccination, cell-based therapies, etc.) against which the tumor in thepatient was identified as being poorly responding, resistant orinsensitive prior to the administration of a BO-11X formulation (i.e.boxes in the bottom of the flowchart).

FIG. 16: Clinical data generated in metastatic lesions in a patient. A46 years-old female with metastatic leiomyosarcoma developed progressivedisease after receiving several lines of treatment (chemotherapy andanti PD-1/anti LAG-3 combination, in particular). She had an abdominalwall cancer lesion that was suitable for injection and with informedconsent received intra-tumoral injections of BO-112. After three dosesof 0.6 mg BO-112, the patient received no further systemic treatment,and only palliative radiation to a femoral (bone) metastasis. (A) Arrowsindicate relevant lesions, showing increase in radiological signs oftumoral necrosis, as established in 4 different locations by imaginganalysis (left column) that was repeated in similar positions 3 monthslater (right column), showing improvement in both BO-112 injected andnon-injected lesions. (B) Before and after BO-112 treatment, populationsof Circulating Immune cells (defined on the basis of specific orcombined cell surface markers such as CD4, CD8, or CD8) were evaluatedin peripheral blood of this patient, indicating that (after an initialreduction) BO-112 treatment may increase the amount of specific cellsubpopulations in circulation, in particular NK cells and CD16-positivecells.

FIG. 17: Clinical data generated in a 72 year-old female patientpresenting metastatic melanoma (stage IV) with progressive disease. Shepreviously underwent multiple surgeries including phalanx amputation andinguinal lymphadenectomies, as well as radiotherapy. Previous systemictreatment included interferon, after pembrolizumab and fotemustintreatments. She was evaluated with a lesion in the inguinal regionmeasuring 9 cm which was suitable for intra-tumoral injection. Withinformed consent, the inguinal lesion was injected under ultrasoundguidance with a single dose 0.6 mg BO-112 and followed by imaginganalysis that was repeated in similar position in the following weeks.One week after this single dose administration, the target lesion haddecreased in size to 8 cm. The patient then was treated with ipilimumab(anti-CTLA4 antibody, 3 mg/kg every 3 weeks for 4 cycles), resulting ina further improvement in cancer lesions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses compositions, including those defined inthe composition claims of PCT/EP2016/078078, defined therein as BO-11X,and exemplified in Examples 1 and 2 thereof therein, wherein X is awhole number. A group of features of BO-11X composition disclosed inPCT/EP2016/078078 is comprised in a BO-112 composition. In particular,the BO-112 composition can be further defined by combining specificfeatures that apply either to the particles that are comprised in theBO-112 composition or to the physical-chemical features that areassociated to the aqueous composition.

A preferred BO-112 composition is an aqueous composition comprisingparticles wherein:

-   -   (i) each of said particles comprises a complex of at least one        double-stranded polyribonucleotide, or a salt or solvate        thereof, and at least one linear polyalkyleneimine, or a salt        and/or solvate thereof, wherein said double-stranded        polyribonucleotide is polyinosinic-polycytidylic acid        [poly(I:C)] and the average molecular weight of said linear        polyalkyleneimine is between 17 and 23 kDa;    -   (ii) at least 90% of said particles has a mono-modal diameter        distribution below 300 nm;    -   (iii) said particles have a z-average diameter of less than or        equal to 200 nm, as measured according to ISO 22412:2017;    -   (iv) said composition contains polyinosinic-polycytidylic acid        [poly(I:C)] at a concentration of at least 0.5 mg/mL;    -   (v) said composition has: a pH of between 2 and 4; and    -   (vi) said composition has a zeta potential between 35 mV and 50        mV, according to ISO 13099.

In a preferred embodiment, a BO-112 composition is a compositionaccording to the foregoing, wherein at least 90% of said particles havea mono-modal diameter distribution between 30 nm and 150 nm.

In a more preferred embodiment, a BO-112 composition is a compositionaccording to the foregoing, wherein at least 40% of the double-strandedpolyribonucleotides comprised in said particles have at least 850 basepairs, and at least 50% of the double-stranded polyribonucleotidescomprised in said particles have between 400 and 5000 base pairs.

In an even more preferred embodiment, a BO-112 composition is acomposition according to the foregoing, wherein said composition has azeta potential comprised between 38 and 45 mV.

In a still more preferred embodiment, a BO-112 composition is acomposition according to the foregoing, wherein said linearpolyalkyleneimine is a water-soluble, linear homo-polyalkyleneimine orhetero-polyalkyleneimine.

In an even still more preferred embodiment, a BO-112 composition is acomposition according to the foregoing, wherein said linearpolyalkyleneimine is a linear polyethyleneimine.

In a yet more preferred embodiment, a BO-112 composition is acomposition according to the foregoing, wherein the polydispersity indexof said particle diameter is inferior to 1.5.

In a furthermore preferred embodiment of the foregoing, a BO-112composition is a composition according to the foregoing, whereinpolyinosinic-polycytidylic acid [poly(I:C)] contains between 5% and 60%of double-stranded polyribonucleotides having less than 400 base pairs,between 15% and 30% of double-stranded polyribonucleotides havingbetween 400 and 850 base, between 10% and 70% of double-strandedpolyribonucleotides having between 850 and 5000 base pairs, and between0% and 10% of double-stranded polyribonucleotides having more than 5000base pairs.

In a furthermore more preferred embodiment, a BO-112 composition is acomposition according to the foregoing, wherein said composition furthercomprises at least one pharmaceutically acceptable carrier, organicsolvent, excipient and/or adjuvant.

In a furthermore still more preferred embodiment, a BO-112 compositionis a composition according to the foregoing, wherein said compositionfurther comprises at least one compound selected from an organiccompound, an inorganic compound, a nucleic acid, an aptamer, a peptideor a protein.

In a furthermore even more preferred embodiment, a BO-112 composition isa composition according to the foregoing, wherein said compositionfurther comprises glucose or mannitol in a concentration of between 1and 10% (weight/volume).

In a furthermore even still more preferred embodiment, a BO-112composition is a composition according to the foregoing, wherein saidcomposition is an aqueous composition that has an osmolality of between200 and 600 mOsm/kg.

In a furthermore much more preferred embodiment, a BO-112 composition isa composition according to the foregoing, wherein:

-   -   (i) each of said particles is formed by making a complex of at        least one double-stranded polyribonucleotide, or a salt or        solvate thereof, and at least one linear polyalkyleneimine, or a        salt and/or solvate thereof, wherein said double-stranded        polyribonucleotide is polyinosinic-polycytidylic acid        [poly(I:C)] and the average molecular weight of said linear        polyalkyleneimine is between 17 and 23 kDa;    -   (ii) at least 90% of said particles has a mono-modal diameter        distribution below 300 nm;    -   (iii) said particles have a z-average diameter of less than or        equal to 200 nm, as measured according to ISO 22412:2017;    -   (iv) said composition contains polyinosinic-polycytidylic acid        [poly(I:C)] at a concentration of at least 0.5 mg/mL;    -   (v) said composition has: a pH of between 2 and 4; and    -   (vi) said composition has a zeta potential equal or superior to        30 mV, according to ISO 13099;    -   wherein said particles are formed at the ratio of the number of        moles of nitrogen of said polyalkyleneimine to the number of        moles of phosphorus of said double-stranded polyribonucleotide        in said composition being equal to or greater than 2.5.

In a preferred embodiment of the furthermore much more preferredembodiment, a BO-112 composition is a composition according to theforegoing, wherein said polyinosinic-polycytidylic acid [poly(I:C)] areformed by annealing:

-   -   (i) polyinosinic acid [poly(I)] preparation containing between        80% and 99% of molecules having less than 400 bases, between 0%        and 20% of molecules having between 400 and 850 bases, between        0% and 5% of molecules having between 850 and 5000 bases, and        between 0% and 5% of molecules having more than 5000 bases; and    -   (ii) polycytidylic acid [poly(C)] preparation containing between        20% and 85% of molecules having less than 400 bases, between 10%        and 40% of molecules having between 400 and 850 bases, between        0% and 50% of molecules having between 850 and 5000 bases, and        between 0% and 5% of molecules having more than 5000 bases.

In a more preferred embodiment of the furthermore much more preferredembodiment and preferred embodiment thereof, a BO-112 composition is acomposition according to the foregoing, wherein said composition isformed by additionally adding glucose or mannitol in a concentration ofbetween 1 and 10% (weight/total volume of said composition).

In a more preferred embodiment of the foregoing, saidpolyinosinic-polycytidylic acid [poly(I:C)] are formed by annealing:

-   -   (i) polycytidylic acid [poly(C)] preparation containing between        20 and 82% of molecules having less than 400 bases, between 15        and 40% having 400-850 bases, between 3 and 50% having 850-5000        bases, and less than 1% of molecules having more than 5000        bases; and    -   (ii) polyinosinic acid [poly(I)] preparation containing between        80 and 99% of molecules having less than 400 bases, between 1        and 20% of molecules having 400-850 bases, between 0 and 5% of        molecules having 850-5000 bases, and less than 1% of molecules        having more than 5000 bases.

More preferably polyinosinic-polycytidylic acid [poly(I:C)] are formedby annealing:

-   -   (i) polycytidylic acid [poly(C)] preparation containing between        33% and 73% of molecules having less than 400 bases, between 20        and 37% of molecules having 400-850 bases, between 5 and 48% of        molecules having 850-5000 bases, and less than 1% of molecules        having more than 5000 bases; and    -   (ii) polyinosinic acid [poly(I)] preparation containing between        81 and 98% of molecules having less than 400 bases, between 6        and 17% of molecules having 400-850 bases, between 0 and 3% of        molecules having 850-5000 bases, and less than 1% of molecules        having more than 5000 bases.

In an even more preferred embodiment of the foregoing, the BO-11X andBO-112 compositions comprise poly(I:C) molecules having a sizedistribution whereby said poly(I:C) contains between 7 and 57% ofmolecules having less than 400 bases, between 20 and 45% of moleculeshaving 400-850 bases, between 20 and 70% of molecules having 850-5000bases, and between 0 and 9% of molecules having more than 5000 bases.More preferably, the BO-11X and BO-112 compositions comprise poly(I:C)molecules having a size distribution whereby said poly(I:C) containsbetween 10 and 30% of molecules having less than 400 bases, between 20and 30% of molecules having 400-850 bases, between 40 and 60% ofmolecules having 850-5000 bases, and between 0 and 5% of moleculeshaving more than 5000 bases. Even more preferably, the BO-11X and BO-112compositions comprise poly(I:C) molecules having a size distributionwhereby said poly(I:C) contains between 11 and 28% of molecules havingless than 400 bases, between 23 and 27% of molecules having 400-850bases, between 42 and 55% of molecules having 850-5000 bases, andbetween 0 and 3% of molecules having more than 5000 bases.

A BO-112 composition is also a composition obtainable by lyophilisationof the aqueous composition according to any of the foregoingembodiments. These lyophilised BO-112 compositions may be thenreconstituted using appropriate solutions to provide formulations thatpresent one or more of features defined above, in particular compositioncomprising particles wherein:

-   -   (i) each of said particles comprises a complex of at least one        double-stranded polyribonucleotide, or a salt or solvate        thereof, and at least one polyalkyleneimine, or a salt and/or        solvate thereof;    -   (ii) at least 95%, or at least 90%, of said particles has a        diameter of less than or equal to 900 nm, preferably, less than        or equal to 500 nm (for example, between 140 and 400 nm or        between 140 and 250 nm); and    -   (iii) said particles have a z-average diameter of less than or        equal to 300 nm, preferably less or equal to 200 nm, more        preferably less than or equal to 150 nm, in particular, as        measured according to ISO 22412:2017.

Furthermore, the present invention also relates to a formulationobtainable by treating a cell or tissue from a subject ex vivo with aBO-11x composition, preferably a BO-112 composition, as defined herein.Preferably, said formulation comprises a cell or tissue from a subjectwhich has been treated ex vivo with a composition as disclosed herein.More preferably, said cell or tissue is a cell or tissue in a samplefrom a subject, such as a blood sample, lymph sample or tissue biopsy.

The present invention also provides means for generating an aqueoussolution of poly(I:C) molecules (already containing or not an excipientsuch as glucose or mannitol) and have appropriate features for beingmixed with aqueous solution of a polyalkyleneimine (such aspolyethyleneimine) for producing the BO-11X formulations. Thepoly(I:C)-containing formulation resulting from mixing these two aqueoussolutions is then maintained as a batch preparation (preferably still asan aqueous solution or in a lyophilized form) or can be directlyprepared in aliquots, each contained in a single-use vials, syringes, orother appropriate container for storage, single use of such aliquots,and/or lyophilisation. BO-11X formulations (in a liquid or lyophilizedform) can be stored at room temperature, at a temperature comprisedbetween 8° C. and 2° C., or at a temperature below 0° C. or below −20°C.

In such preferred embodiment, further compounds (such as one or moreantibody, hormone, peptide, excipient, carrier, inhibitor of anenzymatic activity, chemotherapeutic agent, antibiotic, stabilizingagent, labelling agent, organic solvent, preservatives, carriers, orother drug) can be either added in each of the two aqueous solutions (ifnot altering the correct formation of the particles or any other of thefeatures listed above for BO-11X formulations) prior to their mixing orafter that BO-11X formulation has been produced by mixing the twoaqueous solutions (of double-stranded polyribonucleotide andpolyalkyleneimine). Such additional components that are consequentlyadministered at the same time with BO-11X components can provide acomposition with improvements in the bioavailability, efficacy,pharmacokinetic/pharmacodynamic profiles, stability, metabolization, orother property of pharmaceutical interest that are not observed wheneach of initial BO-11X formulation or the additional component (anothercompound of pharmaceutical interest, for instance) is administeredalone, or each of initial BO-11X formulation or the additional componentare administered separately.

In a further preferred embodiment, the BO-11X formulation is for use asa medicament, such as a pharmaceutical composition that is formulated(e.g. as an injectable, aqueous composition, optionally comprising apharmaceutically acceptable carrier, excipient and/or adjuvant) andadministered for the delivery of double-stranded polyribonucleotides toan organ or a tissue in a healthy state, presenting a disease related toa exogenous pathogenic agent (such a bacteria or a virus), or presentingan alteration due to a cell growth disorder characterized by abnormalgrowth of human or animal cells for instance, due to cancer (that is,involving tumorogenic transformation, metastasis, toxic compound), or agynaecological disorder characterized by abnormal growth of cells of thefemale mammal reproductive organs). Thus, in a preferred embodiment, thepresent invention relates to a method of treatment of a diseasecomprising administering the composition of the present invention to ahuman or animal. In a further preferred embodiment, the presentinvention relates to a method of treatment of a cell growth disordercharacterized by abnormal growth of human or animal cells, as definedherein, comprising administering the composition of the presentinvention to a human or animal.

Preferably, the BO-11X formulation is used in methods for inducing(directly or indirectly) the death of the tumor cell or suppress growthof the tumor cell, at scope of treating, reducing, ameliorating, orpreventing cancer growth, survival, metastasis, epithelial-mesenchymaltransition, immunologic escape or recurrence. More preferably, BO-11Xformulations are used in methods for treating solid tumors, such ascarcinomas, gliomas, melanomas, or sarcomas. In particular, the BO-11Xformulation is administered either systemically or more directly withinor in a location near to the tumor such as at the margin of the tumormass, in the surrounding epithelial cells, lymphatic or blood vessels(e.g. by intratumoral or peritumoral injection), or the abnormallygrowing cells of female mammal reproductive organs. A further preferredembodiment of the present invention relates to a BO-112 composition asdefined in the foregoing, for use as a medicament. A further preferredembodiment of the present invention relates to a BO-112 composition foruse, as defined in the foregoing, wherein said medicament is aninjectable, aqueous composition, optionally comprising apharmaceutically acceptable carrier, excipient and/or adjuvant.

Another embodiment of the present invention relates to a BO-112composition, as defined in the foregoing, for use in treatment of a cellgrowth disorder characterized by abnormal growth of human or animalcells. A preferred embodiment relates to the BO-112 composition for useaccording to the foregoing, wherein said cell growth disorder is canceror a gynaecological disorder characterized by abnormal growth of cellsof the female mammal reproductive organs. A more preferred embodiment ofthe aforementioned embodiment relates to the BO-112 composition for useaccording to the foregoing, wherein the composition is for use forintratumoral or peritumoral injection. Alternatively, a more preferredembodiment of the aforementioned embodiment and preferred embodimentthereof relates to the BO-112 composition for use according to theforegoing, wherein the composition is for use for injection at the levelof skin or of an internal organ or tissue.

Another embodiment of the present invention relates to a BO-11Xcomposition, preferably a BO-112 composition, for use as a vaccine, asdefined in the foregoing. Analogously, the present invention alsorelates to a method of treatment in a subject (patient) of a diseaseusing said vaccine. Preferably said BO-11X or BO-112 composition is foruse as a vaccine in treatment of a cell growth disorder characterized byabnormal growth of human or animal cells, more preferably in treatmentof cancer. Alternatively, said BO-11X or BO-112 composition is for useas an infection-related vaccine.

In some embodiments, the BO-11X formulation is produced according to themanufacturing methods, and then defined structurally and functionally,as described in the Examples as BO-112 formulations. The BO-11Xformulation can further exhibit the biological activities that werecharacterized for BO-110 as described in WO2011003883, namely activationof a family helicase MDA-5 or the level of NOXA expression, incombination with the induction of autophagy in cancer cells or in a cellline derived from cancer cells, preferably from a human origin, albeitto an improved degree. Independently from the process for characterizingthe properties and mechanism of action for BO-110 that is described inWO2011003883, examples of cell lines for validating BO-11X formulationsare human SK-Mel-19, SK-Mel-28, SK-Mel-103 and SK-Mel-147 cells, and themurine B16 cells, said melanoma cell lines presenting an increasedexpression of molecules such as Interferon Beta when exposed to a BO-11Xformulation. Additionally, the BO-11X formulation presents no toxicityat the doses that promote tumor cell death in cancer cells lines againstnormal cells that are used as controls, such as melanocytes or otherskin cells, as well as cells of the immune system, which usuallyrepresent sites of secondary toxicity in cancer treatment. The BO-11Xformulation may also, following the autophagy and apoptosis of cancercells (or any other effect of therapeutic interest that this formulationmay induce in such cells), induce the release of cancer cell antigensthat may act as inducers of a tumor-specific immunological response, inparticular when BO-11X formulation is administered locally to cancerouscells or tumors (e.g. by peritumoral or intratumoral injection,administering BO-11X at the margin of tumor mass, in surroundingepithelial cells, lymphatic or blood vessels, or directly within thetumor mass), with or without the simultaneous or sequentialadministration of another drug or other treatment for same indication.

The BO-11X formulation may, following the administration in locationdistant or independent from tumor mass (for example, by intra-muscularor sub-cutaneous injection, or in by ex vivo administration to cellsobtained from the patient and then re-injected) provide effect oftherapeutic interest upon targeting also cells other than cancer cells(including immune cells or other cells in blood circulation or locatednearby the cancer). Such an effect may be due to the induction of therelease of a tumor-specific effector, or the migration of cells towardscancer cells or within tumors, even when peritumoral or intratumoralinjection is not possible (such as in non-solid tumors such as leukemiaor other haematological cancer).

The present invention also relates to a process to manufacture thecomposition, as disclosed herein, which comprises:

-   -   (i) preparing an aqueous solution of at least one        double-stranded polyribonucleotide, or a salt or solvate        thereof, and an aqueous solution of at least one        polyalkyleneimine, or a salt or solvate thereof, wherein either        or both solutions optionally further comprise a pharmaceutically        acceptable carrier, organic solvent, excipient and/or adjuvant;    -   (ii) independently filtering each solution through a filter        having a pore diameter of less than or equal to 500 nm to form        sterilized solutions;    -   (iii) mixing the resulting sterilized solutions in a mixing        chamber to form an aqueous composition by addition of one of        said solutions into the other solution in said mixing chamber,        optionally by injection, at a rate of greater than or equal to 1        mL/min, or by simultaneous addition of each of said solutions        into said mixing chamber, optionally by injection, at a rate of        greater than or equal to 1 mL/min; and optionally    -   (iv) filtering the resulting aqueous composition through a        filter having a pore diameter of less than or equal to 600 nm to        form a filtrate, or centrifuging the resulting aqueous        composition at greater than or equal to 22480 m/s² to form a        supernatant; and, optionally    -   (v) lyophilising the resulting aqueous composition, filtrate or        supernatant.

Thus, in one preferred embodiment, the present invention may relate to aprocess to manufacture the composition, as disclosed herein, whichcomprises:

-   -   (i) preparing an aqueous solution of at least one        double-stranded polyribonucleotide, or a salt or solvate        thereof, and an aqueous solution of at least one        polyalkyleneimine, or a salt or solvate thereof, wherein either        or both solutions optionally further comprise a pharmaceutically        acceptable carrier, organic solvent, excipient and/or adjuvant;    -   (ii) independently filtering each solution through a filter        having a pore diameter of less than or equal to 500 nm to form        sterilized solutions; and    -   (iii) mixing the resulting sterilized solutions in a mixing        chamber to form an aqueous composition by addition of one of        said solutions into the other solution in said mixing chamber,        optionally by injection, at a rate of greater than or equal to 1        mL/min, or by simultaneous addition of each of said solutions        into said mixing chamber, optionally by injection, at a rate of        greater than or equal to 1 mL/min; and    -   (iv) optionally lyophilising the resulting aqueous composition.

In addition, in another preferred embodiment, the present invention mayrelate to a process to manufacture the composition, as disclosed herein,which comprises:

-   -   (i) preparing an aqueous solution of at least one        double-stranded polyribonucleotide, or a salt or solvate        thereof, and an aqueous solution of at least one        polyalkyleneimine, or a salt or solvate thereof, wherein either        or both solutions optionally further comprise a pharmaceutically        acceptable carrier, organic solvent, excipient and/or adjuvant;    -   (ii) independently filtering each solution through a filter        having a pore diameter of less than or equal to 500 nm to form        sterilized solutions;    -   (iii) mixing the resulting sterilized solutions in a mixing        chamber to form an aqueous composition by addition of one of        said solutions into the other solution in said mixing chamber,        optionally by injection, at a rate of greater than or equal to 1        mL/min, or by simultaneous addition of each of said solutions        into said mixing chamber, optionally by injection, at a rate of        greater than or equal to 1 mL/min; and    -   (iv) filtering the resulting aqueous composition through a        filter having a pore diameter of less than or equal to 600 nm to        form a filtrate, or centrifuging the resulting aqueous        composition at greater than or equal to 22480 m/s² to form a        supernatant; and    -   (v) optionally lyophilising the resulting filtrate or        supernatant.

More preferably, the process of the present invention does not comprisea final step of lyophilisation. In the process (or method) of thepresent invention, said double-stranded polyribonucleotide, saidpolyalkyleneimine and said pharmaceutically acceptable carrier, organicsolvent, excipient and/or adjuvant are as disclosed herein. Sterilizingeach solution to form sterilized solutions takes place by independentlyfiltering said solutions through a filter having a pore diameter of lessthan or equal to 500 nm, preferably by independently filtering saidsolutions through a filter having a pore diameter of less than or equalto 300 nm, more preferably by filtering said solutions through a filterhaving a pore diameter of less than or equal to 200 nm. Preferably themixing of the resulting filtrates takes place through the large-scaleconvective transport of eddies and subsequently through elimination ofconcentration differences through purely diffusive transport. The mixingchamber may be any chamber or vessel in which the mixing of saidsolutions begins, such as a flask, reactor or mixer, and having anyappropriate shape (such as cylindrical, spherical, or other shape thatallows the correct mixing within the chamber) in which the mixing isperformed in a controlled manner within a confined space. Morepreferably, said mixing chamber has a fixed volume of between 0.1 and 20mL (or even bigger volumes such as 25, 50, 100 mL or more, allowing thecontinuous, higher yield of product, especially in GMP conditions),furthermore preferably between 0.2 and 10 mL, much more preferablybetween 0.5 and 8 mL. More preferably, mixing takes place by addition,optionally by injection, at a rate of between 1 mL/min and 2000 mL/min,still more preferably at between 10 and 1000 mL/min, furthermorepreferably at between 20 and 500 mL/min. Optional filtering of theresulting aqueous composition to form or collect a filtrate maysubsequently be performed through a filter having a pore diameter ofless than or equal to 600 nm, preferably not exceeding the diameter of500 nm, more preferably not exceeding the diameter of 400 nm, yet morepreferably not exceeding the diameter of 300 nm. Alternatively, optionalcentrifuging of the resulting aqueous composition to form or collect asupernatant may subsequently be performed at greater than 22480 m/s2(5000 rpm on a rotor having a radius of 0.082 m), preferably at greaterthan 27202 m/s2 (5500 rpm on a rotor having a radius of 0.082 m), morepreferably at greater than 32372 m/s2 (6000 rpm on a rotor having aradius of 0.082 m), yet more preferably 44062 m/s2 (7000 rpm on a rotorhaving a radius of 0.082 m). In one especially preferred embodiment step(iv) is obligatory when the composition of the present invention is notachieved by steps (i) to (iii) of the process of the present invention,namely when addition is carried out at such that a rate that less than95% of the particles comprised in said aqueous composition has adiameter of less than or equal to 600 nm; and/or said particles have az-average diameter of greater than 200 nm, more preferably when saidparticles do not have a mono-modal diameter distribution. This may bethe case when addition is performed at a rate of between 1 mL/min and 20mL/min, particularly in a mixing (reaction) chamber of between 0.5 and20 mL. Finally, the resulting aqueous composition, filtrate orsupernatant may be subjected to lyophilisation to afford the compositionof the present invention as a particulate solid.

Thus, in one much more especially preferred embodiment of the process ofthe present invention, said process comprises

-   -   (i) preparing an aqueous solution of at least one        double-stranded polyribonucleotide, or a salt or solvate        thereof, and an aqueous solution of at least one        polyalkyleneimine, or a salt or solvate thereof, wherein either        or both solutions optionally further comprise a pharmaceutically        acceptable carrier, organic solvent, excipient and/or adjuvant;    -   (ii) independently filtering each solution through a filter        having a pore diameter of less than or equal to 200 nm to form        sterilized solutions;    -   (iii) mixing the resulting sterilized solutions in a mixing        chamber to form an aqueous composition by addition of one of        said solutions into the other solution in said mixing chamber,        optionally by injection, at a rate between 20 mL/min and 100        mL/min, or by simultaneous addition of each of said solutions        into said mixing chamber, optionally by injection, at a rate        between 20 mL/min and 100 mL/min, wherein said mixing chamber        has a volume of between 0.2 and 10 mL;    -   (iv) filtering the resulting aqueous composition through a        filter having a pore diameter of less than or equal to 500 nm to        form a filtrate, or centrifuging the resulting aqueous        composition at greater than or equal to 32372 m/s² (6000 rpm on        a rotor having a radius of 0.082 m) to form a supernatant; and    -   (v) optionally lyophilising the resulting filtrate or        supernatant.

In another even much more especially preferred embodiment of the processof the present invention, said process comprises

-   -   (i) preparing an aqueous solution of at least one        double-stranded polyribonucleotide, or a salt or solvate        thereof, and an aqueous solution of at least one        polyalkyleneimine, or a salt or solvate thereof, wherein either        or both solutions optionally further comprise a pharmaceutically        acceptable carrier, organic solvent, excipient and/or adjuvant;    -   (ii) independently filtering each solution through a filter        having a pore diameter of less than or equal to 200 nm to form        sterilized solutions;    -   (iii) mixing the resulting sterilized solutions in a mixing        chamber to form an aqueous composition by addition of one of        said solutions into the other solution in said mixing chamber,        optionally by injection, at a rate between 30 mL/min and 100        mL/min, or by simultaneous addition of each of said solutions        into said mixing chamber, optionally by injection, at a rate        between 30 mL/min and 100 mL/min, wherein said mixing chamber        has a volume of between 0.5 and 8 mL; and    -   (iv) optionally lyophilising the resulting aqueous composition.

In a further embodiment, the BO-11X formulation is produced according toa manufacturing process that involves the mixing of two aqueoussolutions, a first one comprising the double-strandedpolyribonucleotides (or a salt or solvate thereof) and a second onecomprising the polyalkyleneimine (or a salt or solvate thereof) so thatthe resulting particles present the features defined above, inparticular with respect to the diameter and the mono-modal diameterdistribution as well the appearance as an essentially clear colloidalsolution.

In a further optional step, an additional compound (being animmune-related adjuvant or therapeutic compound, such as CD40L, IL12,viral or tumor antigens, or anti-cancer drug) can be added during step(i), or after of any of the steps (i)-(v) or the steps (i)-(iv) in theprocess to manufacture the composition defined above. The resultingcompositions (that can be named as BO-11Xm and more specifically asBO-112m, when presenting the combination of features as described above)can be used in accordance to any of the embodiments that are hereindisclosed for using BO-11X compositions, and specifically BO-112compositions.

As described in further details in the Examples, the BO-11X formulationscan be provided by filtering and/or centrifuging pharmaceuticalcomposition comprising particles formed by double-strandedpolyribonucleotides and a water-soluble, polycationic homo- orhetero-polymer, providing the BO-11X formulations as bulk or single useliquid compositions without visible aggregates. Such a process tomanufacture the composition comprises:

-   -   (i) preparing an aqueous solution of at least one        double-stranded polyribonucleotide, or a salt or solvate        thereof, and an aqueous solution of at least one        polyalkyleneimine, or a salt or solvate thereof, optionally        together with a pharmaceutically acceptable carrier, organic        solvent, excipient and/or adjuvant present in either solution;    -   (ii) mixing said solutions; and    -   (iii) filtering the resulting mixture through a filter having a        pore diameter of less than or equal to 600 nm, or centrifuging        at greater than 22480 m/s².

This process can be further adapted for the actual components of thecomposition (double-stranded polyribonucleotides, the polyalkyleneimine,and optional further components) as well as the desired methods ofusing, storing, shipping, packaging, and/or administering thecomposition, in particular if the composition requires to bemanufactured immediately prior to the use or, as is more common forpharmaceutical compositions, manufactured for long-term storage and/orin the form of multiple containers each one for a single-use (e.g. insterile vials or syringes), and containing particles having the mostuniform size, poly(I:C) content, stability, solubility, and, finally,biological effects when administered.

The mixing and filtering steps (ii) and (iii) above can be adapted atthe level of order of filtering and/or mixing, method of mixing, themixing speed, and/or the amount of solutions that is mixed. In a furtherpreferred embodiment, the process to manufacture BO-11X formulationscomprises:

-   -   (i) preparing an aqueous solution of at least one        double-stranded polyribonucleotide, or a salt or solvate        thereof, and an aqueous solution of at least one        polyalkyleneimine, or a salt or solvate thereof, optionally        together with a pharmaceutically acceptable carrier, organic        solvent, excipient and/or adjuvant present in either solution;    -   (ii) sterilizing each solution by filtering them independently;    -   (iii) mixing said solutions in the container for storing,        lyophilising, and/or using the composition by adding either        solution first and then adding the other solution at an        injection speed superior to 50 rpm at a flow speed between 1        mL/min and 50 mL/min; and    -   (iv) sealing the container.

In a further optional step, another compound (being an immune-relatedadjuvant or therapeutic compound, such as CD40L, IL12, viral or tumorantigens, or being an adjuvant or therapeutic compound, such ananti-cancer drug) can be added during step (i), or after of any of thesteps (i)-(v) or the steps (i)-(iv) in the process to manufacture thecomposition defined above. The resulting compositions (that can be namedas BO-11Xm and more specifically as BO-112m, when presenting thecombination of features (i)-(vi) as described above) can be used inaccordance to any of the embodiments that are herein disclosed for usingBO-11X compositions, and specifically BO-112 compositions.

Depending on how the aqueous composition of the present invention is tobe administered (e.g. by sub-cutaneous or intratumoral injection), theBO-11X composition or BO-112, as defined in the foregoing, can beprovided in containers and/or in amount that are the most appropriatefor single use or multiple dose.

Combinations

A composition according to the present invention can be used incombinations including other compounds or treatments with which it isknown to be compatible, if not providing an additive or even synergisticeffect. Moreover, the compositions that are be named as BO-11Xm (andspecifically as BO-112m) can be produced as described above providingnew products in which the combination of a BO-11X composition, such as aBO-112 compositions, with another compound provides improved therapeuticeffects or other pharmaceutically relevant effects (including stabilityor efficacy at lower doses, in general or with respect to specificindications or routes of administration) when compared to combinationswherein the BO-11X composition and the other compound are simply mixedbefore use.

For instance, poly(I:C) molecules can be used in combination withdifferent anti-cancer drugs, antibodies, radiotherapy, or chemotherapy(Le U et al., 2008; Le U et al., 2009; Taura M et al., 2010; Matijević Tet al., 2011; Levitzki A, 2012; Yoshino H and Kashiwakura I, 2013;Hafner A et al., 2013) or different length of poly(I:C) molecules (ZhouY et al., 2013). Poly(I:C) molecules have also been used as an adjuvantor synergistiically-acting agent when combined with other agents such asin vaccination with cancer antigens or cell lysates (Ammi R et al.,2015), agents blocking PD-1/PD-L1 pathway (Nagato T and Celis E, 2014),other TLR agonists, such as TLR9 agonist CpG ODN (Zhang Y et al., 2014),dichloroacetate (Ohashi T et al., 2013), IL27 (Chiba Y et al., 2013),kinase inihibitors such as sorafenib (Ho V et al., 2015), proapoptoticproteins such as NS1 (Gupta S et al. 2016), Zoledronic acid (Chen L etal., 2013), or all-trans retinoic acid (Szabo A et al., 2012). Otheruses of BO-11X formulations may become apparent in view of activities ofpoly(I:C) molecules towards specific cell types recently demonstrated,at least using in vitro assays, such as on pre-adipocytes, inhibitingdifferentiation and differentiation in adipocytes (Yu L et al., 2016),mesenchymal stem cells, enhancing immunosuppressive effects (Cho K. etal., 2016; Vega-Letter A et al., 2016), or activation of NK cells(Perrot I et al., 2010).

In some aspects, the present invention relates to a pharmaceuticalcomposition comprising an effective amount of a BO-11X formulation andan effective amount of one or more immune-modulating agents, inparticular agents that target immune checkpoint molecules (such as PD-1,PD-L1, PD-L2, CTLA-4, CD134, CD134L, CD137, CD137L, CD80, CD86, B7-H3,B7-H4, B7RP1, LAG3, ICOS, TIM3, GAL9, CD28, AP2M1, SHP-2, PPP2R5A orOX-40), said compounds commonly named as checkpoint inhibitors (CPIs).Alternatively the immune-modulating agent blocks the immunosuppressiveeffect of T regulatory cells such as an inihibitor of drug targets suchIDO, TGF-β, STAT3, or CSFR1.

Accordingly, the present invention provides compositions and methodsthat are useful in combination therapies and regimens comprising theadministration of BO-11X formulations (or BO-11Xm formulations) andanother therapeutic agent or treatment (including radiotherapy,chemotherapy, cryotherapy, tumor ablation, or photodynamic therapy). Inparticular, the present invention relates to a method for treating,ameliorating, or preventing cancer growth, metastasis, ulceration,immunologic escape or recurrence in a subject, comprising administeringa BO-11X formulation and one or more anticancer drug, preferably animmune-modulating agent, wherein the administration is simultaneous (asseparate formulations or in the context of a co-formulation) orsequential (in any order or in consecutive cycles of administration). Insome aspects, the present invention relates to a method for treatingcancer, comprising administering an effective amount of BO-11Xformulation agent and an effective amount of one or moreimmune-modulating agents to a subject in need thereof, in particularwherein the subject is undergoing cancer therapy with one or moreimmune-modulating agents.

In some embodiments, the immune-modulating agent is preferably anantibody including a monoclonal antibody and other antibody formats, orany other pharmaceutically available agent that binds a cell surfaceprotein that control immune response, thus acting as a CPI, which canblock, reduce and/or inhibit PD-1 and PD-L1 or PD-L2 and/or the bindingof PD-1 with PD-L1 or PD-L2. Alternatively, this CPI can block, reduceand/or inhibit reduces and/or inhibits the activity of other immunecheckpoint molecules such as CTLA-4, AP2M1, CD80, CD86, SHP-2, and/orPPP2R5A As a further alternative, the CPI increases and/or stimulatesCD137 (4-1BB) and/or the binding of CD137 (4-1 BB) with one or more of4-1BB ligand and TRAF2.

In some embodiments, the methods involving combination therapies andregimens for the treatment of cancer comprising the administration ofBO-11X formulations (or BO-11Xm formulations) may further defined withrespect to a specific administration method, wherein the BO-11Xformulation is administered by a route different form the one of theother therapeutic compound, such as an immune-modulating agent, andpreferably a CPI. This method may involve administering the BO-11Xformulation by intratumoral or peritumoral injection (within the tumor,at the margin of the tumor mass, in the surrounding epithelial cells,lymphatic or blood vessels) or other means that allow administering theBO-11X formulation directly within or in proximity of cancer cells ororgan comprising the cancer cells (and not indirectly, for instancethrough bloodstream) and the systemic administration of CPI or otherimmunostimulatory agent). The BO-11X formulation by intratumoral orperitumoral injection may be performed at the level of skin, i.e. intothe skin (e.g. for treating melanoma or in connection to the combinationwith a vaccine) or of an internal organ or tissue, i.e. into saidinternal organ or tissue (e.g. by intrahepatic injection for treatingliver cancer or intravesicular administration for bladder cancer). Suchlocal administration of BO-11X formulation preferably follows or(preferably) is followed by the administration of the immunostimulatoryagent.

The BO-11X formulation (or a BO-11Xm formulation) can be alsoadministered in combination to cell-based therapies, wherein the BO-11Xis either co-administered with the cell-based therapy directly to thepatient, or is used for treating cells that are obtained from a patient(from the blood or from a biopsy including cells twithin the tumor mass,at the margin of tumor mass, in surrounding epithelial cells, lymphaticor blood vessels). These cells, with or without positive or negativeselection for specific cell types, can be exposed to BO-11X formulationin an appropriate laboratory setting for generating cells that,following this treatment, present markers, secrete proteins and/orexpose antigens useful for further cancer treatment. Such autologouscells, again with or without further negative or positive selection, canbe administered to the patient. During later phases of the treatment,BO-11X formulation can be further administered to the patient by anyappropriate mean (by intratumoral or, preferably, intra-muscular orsub-cutaneous injection). The ex vivo treatment of cells from patientscan be performed for a period of time of greater than 1 hour, preferablygreater than 3 hrs, more preferably greater than 8 hrs, even morepreferably greater than 24 hrs, or more, at a concentration that lowerto the one intra-muscular or sub-cutaneous injection (preferably at 50%,more preferably at 25%, even more preferably at 10%, furthermorepreferably at 5% or much more preferably at 1% of such dose).

Additional Effects and Uses

In a further embodiment, the present invention provides pharmaceuticaluses and methods involving the administration of a BO-11X formulation(or a BO-11Xm formulation) for increasing immune response against apathogen or undesirable biological agent and in particular for enhancingan anti-tumor immune response, potentially acting itself as animmune-modulating agent. Such an effect can be monitored by measuringtumor-related immune response into the tumor site and tumormicroenvironment (or in the bloodstream, other biological fluids, andtissues) at the level of relevant cell types or subpopulations (e.g.dendritic cells, T regulatory cells, T cells and/or NK cells) and/or ofimmunological biomarkers (e.g. chemokines, growth factors, cytokines,and their receptors).

In particular, when the BO-11X formulation (or a BO-11Xm formulation) isclinically administered by intratumoral injection, apoptosis and/ornecrosis is observed in the tumor, thus potentially promoting thepresentation of tumor antigens to resident dendritic cells. A signalingcascade may lead also to the recruitment of immune cells, in particularCD4+ and CD8+ T cells into the tumor mass promoting an immune effectagainst the tumor, contributing to the cytotoxic effect of BO-11X.

Another embodiment relates to a method for inducing interferonproduction in a cell or tissue in vitro, comprising the step ofcontacting a cell or tissue with a composition, wherein said cell ortissue exhibits a cell growth disorder characterized by abnormal growthof human or animal cells, wherein said composition is BO-11X, morepreferably BO-112. Thus, the invention relates to a method for inducinginterferon production in cells from a subject in vitro, comprising:

-   -   (i) obtaining the cells from the subject;    -   (ii) contacting said cells with a composition of BO11X,        preferably BO-112.

Pharmaceutical Compositions

Also provided are methods for making a pharmaceutical composition ofBO-112 (and/or other BO-11X formulations, such as a BO-11Xm or BO-112mformulation) by mixing a BO-11X formulation and one or morepharmaceutically acceptable adjuvant, diluent, carrier, or excipientthereof. Such components can be adapted for the specific medicalindication (e.g. a solid cancer or a haematological cancer) and/or theadministration means e.g. by injection (peri-tumoral, intra-tumoral,intra-hepatic, intra-pancreatic, intra-muscular, or sub-cutaneousinjection), by inhalation, topically, or orally.

BO-11X formulation-based combination treatments may involve the same ordifferent administration routes for BO-11X formulation and the othercompound. In particular, the BO-11X formulation can be administered, asfor other immunostimulatory RNA agents, by intratumoral or peritumoralinjections that may activate the immune system prior to the systemicadministration of a therapeutic antibody acting as CPI such a PD-1/PD-L1pathway inhibitors (Bald T et al., 2014) or activated T cells (Amos S Met al., 2011). Alternatively, the BO-11X formulation together with othertherapeutic compounds being TLR agonist or ligands such as CpG moleculesor Resiquimod for enhancing the effect of anticancer vaccination(Sajadian A et al., 2014) or dendritic cells (Fujimura T et al., 2006).

Immune-Modulating Agents

The BO-11X formulation (including a BO-11Xm formulation) may be combinedwith one or more immune-modulating agents. In some embodiments, theimmune-modulating agent is a co-stimulatory or co-inhibitory molecule(e.g. of one or more immune cells, such as, by way of non-limitation, Tcells and NK cells). In some embodiments, the immune-modulating agent isan agent that modulates a CD4 and/or CD8 T cell, for instance by actingas agonist or antagonist with respect to CD3, CD4, CD8, PD-1, PD-L1,PD-L2, CTLA-4, CD137, CD96, CD73, CD90, CD47, CD69, CD26, TIM3, andLAG3. In other embodiments, the immune-modulating agent is an agent thatmodulates NK cells, for instance by acting as agonist or antagonist withrespect to CD3, NKp46, CD16, NKG2D, NKp44, and NKp30. In otherembodiments, the immune-modulating agent is an agent that modulatestumor stroma and endothelium biomarkers, for instance by acting asagonist or antagonist with respect to CD45, PD-L1, PD-L2, PTEN, andCD31, or combinations thereof. Combination of immune-modulating agentsmay be used to target different immune populations, whereby the combinedagents target cells expressing different immune cell populations.

The immune-modulating agent is provided as a further compound in form ofa chemical organic or inorganic compound, a nucleic acid, an aptamer, apeptide, a protein, and more particularly an antibody that binds therelevant target in the biological fluids or on cell surface. Theantibody may be polyclonal or monoclonal; intact or truncated (e.g.,F(ab′)₂, Fab, Fv); bispecific or multispecific; xenogeneic, allogeneic,syngeneic, or modified forms thereof (e.g., a chimeric antibody or ahumanized antibody). When the immune-modulating agent is a monoclonalantibody, it may be a non-human mammal-derived monoclonal antibody, arecombinant chimeric monoclonal antibody, a recombinant humanizedmonoclonal antibody, or a human monoclonal antibody.

The antibody that acts as immune-modulating agent can further comprisethe structural elements normally required for exerting the requiredbiological activity, such as four polypeptide chains, two heavy (H)chains and two light (L) chains inter-connected by disulphide bonds thatare capable of binding one or more antigens (e.g. bi-specific ormulti-specific antibodies) and presenting an Fc region of animmunoglobulin (e.g. IgA, IgG, IgE, IgD or IgM) which may interact withFc receptors and activate an immune response leading to depletion and/orcell death of immune cells or other cells. Each heavy chain is comprisedof a heavy chain variable region (V_(H)) and a heavy chain constantregion. The heavy chain constant region is comprised of three domains,CH₁, CH₂ and CH₃. Each light chain is comprised of a light chainvariable region (V_(L)) and a light chain constant region. The lightchain constant region is comprised of one domain, CL. The V_(H) andV_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FR). Each variable region (V_(H) or V_(L)) contains 3 CDRs,designated CDR1, CDR2 and CDR3. Each variable region also contains 4framework sub-regions, designated FR1, FR2, FR3 and FR4. The termantibody includes all types of antibodies, including, for example, IgA,IgG, IgD, IgE and IgM, and their respective subclasses (isotypes), e.g.,IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. An antibody, in someembodiments, also refers to antibody fragments and antigen-bindingfragments.

Antibodies suitable for practicing the methods described herein can beof various antibody formats, for example, monoclonal, polyclonal,bispecific, multispecific, and can include, but are not limited to,human, humanized or chimeric antibodies, comprising single chainantibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fabexpression library, and/or binding fragments of any of the above.Antibodies also refer to immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain at least two antigens or target binding sites against at leasttwo targets described herein. The immunoglobulin molecules describedherein can be of any type (e.g. IgG, IgE, IgM, IgD, IgA and IgY), class(e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. In addition, antibodies (e.g. mono-, bi-,and/or multi-specific) suitable for practicing the invention describedherein can be provided in any of the alternative formats that aredisclosed in the literature, for example Diabodies; Flexibodies; CamelidAntibodies, Immunobodies; Triomabs, Pepbodies, Vaccibodies, minibodies,Fcabs, UniBodies, or DuoBodies (Storz U, 2011).

PD-1 (also known as CD279 or Programmed cell death protein 1) is amember of the B7 family of receptors. In some embodiments, PD-1 refersto the human PD-1 sequence (see, e.g. NCBI Reference Sequence:NP_005009) and any naturally occurring allelic, splice variants, andprocessed forms thereof (Keir Metal., 2008; UniProt: Q15116). PD-1 bindsPD-L1 (also known as CD274 or B7-H1) and PD-L2 (also known as CD273 orB7-DC), which are also members of the B7 family. In some embodiments,PD-L1 refers to human PD-L1 (see, e.g. GenBank: AF233516), PD-L2 refersto human PD-L2 (e.g. NCBI Reference Sequence: NM_025239), together withand any naturally occurring allelic, splice variants, and processedforms thereof.

In some embodiments, the immune-modulating agent targets one or more ofPD-1, PD-L1, and PD-L2. In particular, the immune-modulating agent isPD-1 inhibitor. In some embodiments, the immune-modulating agent is anantibody specific for one or more of PD-1, PD-L1, and PD-L2. Suchimmune-modulating agent is an antibody such as, by way ofnon-limitation, nivolumab, (ONO-4538/BMS-936558, MDX1106, OPDIVO),pembrolizumab (KEYTRUDA), pidilizumab (CT-011), MK-3475, BMS 936559,MPDL3280A, and others recently reviewed (Tan S et al. 2016).

In some embodiments, the BO-11X formulation (or a BO-11Xm formulation)is combined with one or more of BMS-936559 and MED14736 for treatmentof, for example, advanced solid tumors. In some embodiments, the BO-11Xformulation is combined with one or more MPDL3280A (optionally withvemurafenib) and MED14736 (optionally with one or more of dabrafenib andtrametinib) for the treatment of melanoma. In some embodiments, theBO-11X formulation is combined with one or more MPDL3280A (optionallywith erlotinib) and MED14736 (optionally with tremelimumab) for thetreatment of NSCLC. In some embodiments, the BO-11X formulation iscombined with MPDL3280A (optionally with one or more of bevacizumab andsunitinib) for the treatment of RCC. In some embodiments, the BO-11Xformulation is combined with MPDL3280A for the treatment of solid orhematological malignancies. In some embodiments, the BO-11X formulationis combined with one or more MPDL3280A (optionally with one or more ofbevacizumab, chemotherapy and cobimetinib); MED14736 (optionally withtremelimumab) and MSB0010718C for the treatment of solid tumors. In someembodiments, the BO-11X formulation is combined with AMP-224 for thetreatment of advanced cancer. In some embodiments, the BO-11Xformulation is combined with nivolumab (optionally with iliolumbar(anti-KIR)) for the treatment of advanced solid tumors. In someembodiments, the BO-11X formulation is combined with nivolumab for thetreatment of castration-resistant prostate cancer, melanoma, NSCLC, andRCC. In some embodiments, the BO-11X formulation is combined withpembrolizumab for the treatment of colon cancer. In some embodiments,the BO-11X formulation is combined with pembrolizumab for the treatmentof gastric cancer, head and neck cancer, TNBC, and urothelial cancer. Insome embodiments, the BO-11X formulation is combined with nivolumab(optionally with ipilimumab) for the treatment of gastric cancer,pancreatic cancer, small-cell lung cancer, and TNBC. In someembodiments, the BO-11X formulation is combined with nivolumab(optionally with ipilimumab) for the treatment of glioblastoma. In someembodiments, the BO-11X formulation is combined with nivolumab for thetreatment of hepatocellular cancer. In some embodiments, the BO-11Xformulation is combined with pembrolizumab for the treatment of Hodgkinlymphoma, myeloma, myelodysplastic syndrome, and non-Hodgkin lymphoma.In some embodiments, the BO-11X formulation is combined with pidilizumabfor the treatment of malignant gliomas. In some embodiments, the BO-11Xformulation is combined with one or more of nivolumab (optionally withone or more of ipilimumab, and multiple class 1 peptides and montanideISA 51 VG; and optionally sequentially with ipilimumab) andpembrolizumab for the treatment of melanoma. In some embodiments, theBO-11X formulation is combined with pembrolizumab for the treatment ofmelanoma and NSCLC. In some embodiments, the BO-11X formulation iscombined with one or more of nivolumab (optionally with one or more ofgemcitabine/cisplatin, pemetrexed/cisplatin, carboplatin/paclitaxel,bevacizumab, erlotinib, and ipilimumab) and pembrolizumab for thetreatment of NSCLC. In some embodiments, the BO-11X formulation iscombined with pidilizumab (optionally with gemcitabine) for thetreatment of pancreatic cancer. In some embodiments, the BO-11Xformulation is combined with pidilizumab (optionally with one or more ofsipuleucel-T and cyclophosphamide) for the treatment of prostate cancer.In some embodiments, the BO-11X formulation is combined with one or moreof nivolumab (optionally with one or more of sunitinib, pazopanib, andipilimumab), pembrolizumab (optionally with pazopanib), and pidilizumab(optionally with dendritic cell/RCC fusion cell vaccine) for thetreatment of RCC. In some embodiments, the BO-11X formulation iscombined with one or more of anti-LAG3 (BMS-986016, optionally withnivolumab), nivolumab (optionally with interleukin-21), and AMP-554 forthe treatment of solid tumors. In some embodiments, the BO-11Xformulation is combined with pembrolizumab for the treatment of solidtumors.

In some embodiments, the immune-modulating agent targets one or more ofCD137 or CD137L. In some embodiments, the immune-modulating agent is anantibody specific for one or more of CD137 or CD137L. For instance, insome embodiments, the immune-modulating agent is an antibody such as, byway of non-limitation, urelumab (also known as BMS-663513 and anti-4-1BBantibody). In some embodiments, the BO-11X formulation is combined withurelumab (optionally with one or more of nivolumab, lirilumab, andurelumab) for the treatment of solid tumors and/or B-cell non-Hodgkinslymphoma and/or head and neck cancer and/or multiple myeloma. In someembodiments, the immune-modulating agent is an antibody such as, by wayof non-limitation, ipilimumab (MDX-010, MDX-101, Yervoy, BMS) and/ortremelimumab (Pfizer). In some embodiments, the BO-11X formulation iscombined with ipilimumab (optionally with bavituximab) for the treatmentof one or more of melanoma, prostate cancer, and lung cancer.

In some embodiments, the immune-modulating agent targets CD20. In someembodiments, the immune-modulating agent is an antibody specific CD20.For instance, in some embodiments, the immune-modulating agent is anantibody such as, by way of non-limitation, Ofatumumab, obinutuzumab(GAZYVA), AME-133v, Ocrelizumab, TRU-015, and veltuzumab.

Validation of BO-11X Formulations

The pre-clinical validation of therapeutic efficacy of a BO-11Xformulation (in accordance to the present invention, including a BO-11Xmformulation, and as exemplified by BO-112 in the Examples) can beperformed in cell-based assays and, most interestingly, in animal modelswhere different experimental criteria can be tested and compared toestablish the most appropriate conditions to achieve therapeutic effectseffectively, using the BO-11X formulation alone or in combination with acandidate or approved anti-cancer drug, These criteria include thedoses, administration route, the order and/or the frequency ofadministration of either compound at the scope to identify which are thebetter conditions for therapeutic use of a BO-11X formulation (alone, asa BO-11Xm formulation, or synergistically with a candidate or approvedanti-cancer drug) in terms of efficacy, safety, and/or clinical use.When using animal and/or ex vivo models, BO-11X formulations can bevalidated using one or more of the following criteria: cancer cellcytotoxicity, PARP activation, immunogenic cell death, therapeuticefficacy (in particular through apoptosis and/or necrosis of cancercells, either directly or indirectly by abscopal effect), increasedexpression of markers on immune cell populations, increased populationsof immune cells, and the other assays shown in the Examples.

The effects of different dosages of a BO-11X formulation (or a BO-11Xmformulation), number of and/or site of administration (in particular, byinjecting it in one or more sites), each compound, route ofadministration, frequency, and/or time point for administration can beassociated to relevant end-points and physiological parameters that aremeasured in biological samples obtained from cells or (preferably)animals that are exposed to the tested compounds, alone or incombination with other drugs. A non-limiting list of such parametersincludes regression of tumor size, block of tumor growth and/orproliferation of tumor cells, apoptosis, reduced tumor vascularizationor metastasis, overcoming resistance to a common anti-cancer drug (orotherwise improving the response to such a drug in the treatedpopulation), reduced treatment related-adverse events on normal tissuesand functions, modulation of immune response and/or of immune cellshaving specific activities and features, identification of biomarkers orspecific cell populations in biological materials (e.g. present incancer cell preparations, tumor biopsies or biological fluids) whoseincrease (or decrease) is known in the literature as being associated toanti-cancer effect in general, and in particular to survival of animalmodels and possibly of cancer patients. Whenever possible, suchend-points are measured at intermediate and final time-points followingthe administration of each test compound, or a test combination ofcompounds at a given dose and/or regimen, by using a specific route ofadministration and/or pharmaceutical formulations.

The therapeutic, anti-cancer efficacy of BO-11X formulation (or BO-11Xmformulations) can be tested alone or in combination withstandard-of-care, conventional treatments (such as radiotherapy,chemotherapy, inhibitors of cellular kinases, drugs having epigeneticeffects, etc.) or treatments involving novel mechanisms and/or novelcandidate anti-cancer drugs. Indeed, a category of novel anti-cancercompounds that can be tested in combination with a BO-11X formulationare those improving the anti-tumor immune responses within tumormicroenvironment, by providing a systemic antitumor immunity thattargets disseminated tumor cells are eliminated. This approach has beenproven successful in animal models and patients and can improve theoutcome of conventional therapies such as radiotherapy or chemotherapy(see, for example, Galluzzi L et al., 2014; Vacchelli E et al., 2013;Van der Jeught K et al., 2015).

This growing panel of new cancer drugs targets the mechanisms by whichcancer cells escape from immune detection and destruction by human body.Cancer-specific immunotherapies may provide a series of advantages whencompared to other cancer therapies (e.g. tumor cell specificity).Indeed, the identification of a series of molecular targets for cancerimmunotherapies is allowing major advances in defining the mechanismsand compounds that can provide an appropriate co-stimulatory (orco-inhibitory) effect on immune responses against tumors with respect totheir plasticity, heterogeneity, resistance, or microenvironment.

A non-limiting list of cancer passive or active immunotherapies, eachacting on different molecular targets and/or mechanisms, includestumor-targeting or other immunomodulatory monoclonal antibodies,oncolytic viruses, immunostimulatory cytokines, adoptive cell transferand other cell-based therapies, DNA- or peptide-based vaccines,inhibitors of immunosuppressive metabolism, or agonists of patternrecognition receptors. Among these mechanisms, molecular targets againstwhich antibodies or other compounds having either agonistic orantagonistic activity (depending on their role in immune response orcancer escape from immune response) include a series of cell membraneproteins such as PD-1, PD-L1, PD-L2, CTLA-4, CD137, CD38, OX-40, CD26,TIM3, and LAG3 that are checkpoints for tumor development and againstwhich different agonistic or antagonistic antibodies are availablecommercially or in scientific repositories for characterizing theirspecificity and/or level of anti-cancer effect against the human antigenor (when dealing with animal models) the corresponding rodent antigen.

Despite impressive patient responses to agents targeting theseco-stimulatory or co-inhibitory molecules (e.g. therapies that are basedon an anti-PD-1 or anti-PD-L1 antibody) the clinical response to immunecheckpoint inhibitors is still incompletely or inefficiently achievingthe desired therapeutic effect in too many cancer patients. A BO-11Xformulation in an appropriate combination with a compound such as ananti-PD-1, anti-CTLA4, or anti-CD137 antibody may enhance the reductionof tumor growth and metastasis (or increase the number of subjectspresenting such reduction), when compared to the effect of theadministration of one of those two anti-cancer agents alone, possiblybeyond the additive effects that may be expected.

The therapeutic effects of BO-11X formulations (or BO-11Xm formulations)can be evaluated in one of the several cell-based models that are basedon isolated or mixed cell culture including primary cancer cells orestablished cancer cell lines, preferably from a human origin.

Examples of cancer cell lines for validating BO-11X formulations can bedefined according to cancer type such as melanoma (human SK-Mel-19,SK-Mel-103 and UACC62 cells; murine B16 cells), carcinoma (mouse Hepa1-6 cells; rat FAO cells), breast cancer (human BT483, HCC1143, HCC1937,MDA-MB-231, MDA-MB-415, MDA-MB-468, and BT549 cells), pancreatic cancer(human MiaPaCa2, IMIM-PC2, Panc1, Panc0203, Panc 3.27, or BxPc3 cells),or other relevant cell lines that are available through ATCC, otherofficial or academic repositories, or commercial providers. Theanticancer effects of BO-11X formulations can be evaluated using aplurality of metrics, including period of time, frequency, and/or dosethat is required to have a block of proliferation, the death, theexpression of biomarkers, and/or the release of signaling molecules(such as chemokines or Interferon Beta) that indicate a potentiallyrelevant effect of the BO-11X formulation to be confirmed under avariety of physiological conditions.

Accordingly, the effects of BO-11X formulations (or BO-11Xmformulations) can be evaluated in tumor animal models in which theanti-tumor response due to the administration of an exemplaryformulation such as BO-112 is assessed in different protocols for bothmonotherapy and combination treatment (e.g. together with a CPI such asan anti-PD-1 antibody) throughout a shorter or longer period of timeafter administration. The study may be pursued by administering BO-112and/or anti-PD-1 antibody in animals at a given time of tumordevelopment due to proliferation of injected cells, that is after aspecific number of days following the injection of cancer cells or(preferably) that present the desired tumor size (e.g. an average sizeof 80-100 mm3), or even following its disappearance (for evaluate anyeffect of each drug or drug combination on tumor relapse). The studywould also involve control compounds that are either negative (e.g.vehicle alone) or positive controls, such as chemotherapeutic or otheranti-cancer drugs that are indicated in the literature as standard fordrug effectiveness for a specific tumor and/or in a given animal model.These activities of validation in animal models and animal cell may alsolead to the development of BO-11X formulation for veterinary use.

The animal model is typically a mouse model in which the cancer appearsfollowing either the transfer and engraftment of human cancer cells(derived from an immortalized cell line or a cancer biopsy that isobtained from a patient) or the induction (or transfer) of mouse tumorcells in the animals. Cells can originated from different types of tumor(e.g. lung carcinoma, melanoma, or lymphoma) and can be injectedsub-cutaneously in the flank of each respective mouse for the detectionof a tumor and for analyzing tumor size and/or composition throughoutthe study. Mice are then treated by randomizing them into groups each ofa size allowing statistical analysis of results (e.g. 3, 5, 10, 12, 15,20 or more animals for each control or treatment group).

Possible improvements afforded by that a BO-11X formulation such asBO-112 and a checkpoint inhibitor such as an anti-PD-1 antibody mayinduce improvements in cancer animal models (in particular whenappropriately combined in terms of amount, order, or otheradministration criteria), increased animal survival after treatmentand/or tumor disappearance, reduced tumor relapse, limited or delayedtoxicity and/or resistance effects, and an improvement in the responseto re-challenge of tumor inoculation after termination of the treatmentwith BO-112 and/or anti-PD-1 antibody.

The exemplary BO-11X formulation that is identified structurally andfunctionally above as BO-112 formulation and an anti-PD-1 antibody canbe administered (alone or in combination, in single or multiple doses)at different locations with respect to tumor cells and/or in differentamount. Typically, BO-112 and the monoclonal antibody specific for mousePD-1 (e.g. clone RMP1-14 from BioLegend or similar ones available fromother providers) are injected sub-cutaneously, intravesicularly,intraperitoneally, peritumorally or intratumorally (depending on themodel and tumor molecular and pathological features) at a concentrationthat is determined with respect to animal weight (e.g. between 0.01 and2.5 mg/kg), concentration in the injected volume (e.g. between 0.01 and0.5 mL, and/or content in each dose (e.g. between 0.01 and 250 μg perdose). In particular, the dose-response of BO-112 at differentconcentrations, combined with a fixed anti-PD-1 antibody dose (or viceversa), may permit determination of any advantageous effect followingthe significant reduction in the amount of either compound that isadministered due to the combination with the other compound (e.g.unaffected or even improved efficacy and/or safety profile; abscopaleffects in tumors that are in different, untreated locations).

BO-112 composition (or a BO-112m composition) can be injected in one ormultiple cycles (e.g. 2, 3, 4, or more) that are separated by a fixed,desired number of days (1, 2, 3, 5, 7, or more). Alternatively, whenBO-112 is co-administered with the anti-PD1 antibody, BO-112 can beinjected immediately before (or after) the antibody (or in a singleinjectable preparation), again in one or multiple cycles (that areseparated by given number of days. Still alternatively, the two agentsmay be formulated, or administered in any sequential order, butseparated by a variable period of time (e.g. 1 hour, 3 hours, 6 hours,12 hours, 18 hours, 24 hours, 36 hours, 2 days or more). In particular,when BO-112 is administered after the anti-PD-1 antibody, its subsequentadministration (alone or further in combination with the anti-PD-1antibody) may provide an anti-tumor rescue effect in animals in whichanti-PD-1 antibody was ineffective against tumor cells, overcoming anyspecific tumor resistance or escape mechanism. At the end of the periodof treatment, all surviving animals can be left untreated for 1, 2, 3,or more consecutive weeks to monitor if and how tumors reappear, with orwithout re-challenging animals that had a complete regression of tumorwith a further sub-cutaneous injection of cancer cells.

The effects of BO-112 compositions (or a BO-112m compositions), alone orin combination with the anti-PD1 antibody as described above, can beassessed as interim results that are reported during the study withoutsacrificing the animals (e.g. by measuring tumor size, percentage ofmice still alive, bodyweight, or behavioural criteria) or aftersacrificing the animal (or in already dead mice) for determiningmolecular features of tumor and/or normal cells (including total numberand/or specific sub-populations of NK cells, tumor-infiltratedlymphocytes, splenocytes, incorporation of radiolabeled precursors, andother cells that may be involved in the anti-tumor local or systemicimmune responses, such as. Myeloid-derived suppressor cells (MDSCs),regulatory T cells (Tregs); tumor associated neutrophils (TANs), M2macrophages, and tumor associated macrophages (TAMs). I parallel, thepresence/absence of relevant biomarkers can be determined by PCRamplification of relevant RNAs, or at protein level on the surface ofcells within tissues or circulating in blood, such as cytokines orchemokines by using standard immunological assays and kits.

This global phenotype analysis can be performed by using cells isolatedfrom tumors, blood, spleen, lymph nodes, or other relevant tissues andlocations, for detecting any statistically and/or therapeuticallyrelevant change in the number of cells expressing cell surface markersthat are detected by flow cytometry and described in the literature suchas CD3, CD4, CD25, FoxP3, CD8, PD-1, PD-L1, PD-L2, PTEN, CTLA-4, CD137,CD96, CD73, CD90, CD47, CD69, CD26, TIM3, LAG3, Gr1, CD11b, Ly6C, Ly6G,NKp46, CD16, NKG2D, NKp44, NKp30, CD45, and CD31. Such cells can be alsoevaluated at the level of tumor antigen-specific immune response,expression of relevant transcription factors, cytokines or chemokines(e.g. IFN-gamma, IFN-beta, TNFalpha, HIF1a, HIF2a, p53), or by usingother cell-based assays.

Additionally, macroscopic examination of organs and skin andmicroscopic, pathological analysis in either immune-deficient fullyimmune competent animal models can further indicate about the efficacyof the study compounds (alone or in the combination) or of theirtoxicity, such as organ inflammation and necropsy. The quantitative datathat are generated in similar studies can be compared among thedifferent experimental groups by using the appropriate statisticaltests, with and without corrections for multiple testing, at the scopeto evaluate which therapeutic (in particular anti-tumor) effects areprovided by the administration of a BO-11X formulation, alone or incombination with another anti-cancer agent.

Methods of Treatment and Patient Selections

In some embodiments, the present invention relates to a method fortreating, reducing, ameliorating, or preventing cancer growth, survival,metastasis, epithelial-mesenchymal transition, immunologic escape orrecurrence, comprising administering by administering a BO-11Xformulation (or a BO-11Xm formulation) and one or more immune-modulatingagents. The cancer may be an oncological disease. The cancer may be adormant tumor, which may result from the metastasis of a cancer. Thedormant tumor may also be left over from surgical removal of a tumor.The cancer recurrence may, for example, be tumor regrowth, a lungmetastasis, or a liver metastasis.

In some embodiments, the cancer is one or more of basal cell carcinoma,biliary tract cancer; bladder cancer; bone cancer; brain and centralnervous system cancer; breast cancer; cancer of the peritoneum;choriocarcinoma; connective tissue cancer; cancer of the digestivesystem (including esophageal, stomach, colon, rectal or othergastrointestinal cancer); eye cancer; cancer of the head and neck;glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm;kidney, adrenal, or renal cancer; leukemia; liver cancer; lung cancer(e.g. small-cell lung cancer, non-small cell lung cancer, lungadenocarcinoma, and lung squamous carcinoma); melanoma; myeloma;neuroblastoma; oral cavity cancer (lip, larynx, tongue, mouth, andpharynx); pancreatic cancer; prostate cancer; retinoblastoma;rhabdomyosarcoma; cancer of the respiratory system; salivary glandcarcinoma; skin cancer; squamous cell cancer; testicular cancer; thyroidcancer; uterine, endometrial, cervical, vulval, ovarian or othergynecological cancer; cancer of the urinary system; lymphoma includingB-cell lymphoma, Hodgkin's and non-Hodgkin's lymphoma (NHL; includingspecific types such as low grade/follicular, small lymphocytic,intermediate grade/follicular, intermediate grade diffuse, high gradeimmunoblastic, high grade lymphoblastic, high grade small non-cleavedcell, or bulky disease NHL), mantle cell and AIDS-related lymphoma;chronic lymphocytic leukemia; acute lymphoblastic leukemia; Hairy cellleukemia; chronic myeloblastic leukemia; as well as other carcinomas andsarcomas; post-transplant lymphoproliferative disorder (PTLD), as wellas abnormal vascular proliferation associated with phakomatoses or edema(such as those that associated with brain tumors). In some embodiments,the cancer is a biliary tract cancer. In some embodiments, the biliarytract cancer is selected from pancreatic cancer, gallbladder cancer,bile duct cancer, and cancer of the ampulla of Vater. In someembodiments, the cancer is liver cancer. In some embodiments, the canceris colon cancer. In some embodiments, the biliary tract cancer ischolangiocarcinoma and/or an adenocarcinoma. Alternatively, the cancermay be listed among the list of rare diseases, being defined accordingto the critera of incidence as defined in Europe or USA, and Indicatedin regularly updated lists made available in the website oforganisations such as the International Rare Cancers Initiative(http://www.irci.info) or RaraCare (http://www.rarecare.eu).

In some embodiments the BO-11X formulation (or a BO-11Xm formulation)and/or immune-modulating agent is used to treat cancers at variousstages (e.g. Stage I, or II, or III, or IV). By way of non-limitingexample, using the overall stage grouping, Stage I cancers are localizedto one part of the body; Stage II cancers are locally advanced, as areStage III cancers. Whether a cancer is designated as Stage II or StageIII can depend on the specific type of cancer. In one non-limitingexample, Hodgkin's disease, Stage II indicates affected lymph nodes ononly one side of the diaphragm, whereas Stage III indicates affectedlymph nodes above and below the diaphragm. The specific criteria forStages II and III therefore differ according to diagnosis. Stage IVcancers have often metastasized, or spread to other organs or throughoutthe body.

In some embodiments, the BO-11X formulations (or BO-11Xm formulations)and/or the immune-modulating agent reduces side effects of the therapiesthat a patient may experience. For example, the combination therapy ofan BO-11X formulation and one or more immune-modulating agent may allowfor a lower dose of the BO-11X formulation and/or one or moreimmune-modulating agent (e.g. as compared to monotherapy) and therebyincrease the therapeutic window of either agent. In some embodiments,the lowered dose mitigates one or more side effects without (or minimal)loss of efficacy. In some embodiments, the BO-11X formulation and/orimmune-modulating agent is used to treat a subject that has atreatment-refractory cancer. In some embodiments, the BO-11X formulationis used to treat a subject that is refractory to one or moreimmune-modulating agents, in particular the one that is actuallycombined with the BO-11X formulation.

In some embodiments (see also Example 3), the subject is refractory to aPD-1 and/or PD-L1 and/or PD-L2 agent, including, for example, nivolumab(ONO-4538/BMS-936558, MDX1106, or OPDIVO), pembrolizumab (KEYTRUDA),pidilizumab (CT-011), MK-3475, BMS-936559, Ibrutinib, and/orMPDL328OA-refractory patients. For instance, in some embodiments, thesubject is refractory to an anti-CTLA-4 agent, e.g. ipilimumab(Yervoy)-refractory patients (e.g. melanoma patients). In someembodiments, the subject is refractory to a BO-11X formulation.Accordingly, in some embodiments the present invention provides methodsof cancer treatment that rescue patients that are non-responsive tovarious therapies, including monotherapy of an BO-11X formulation or oneor more immune-modulating agents.

In particular, certain types of cancer may be selected for the treatmentwith a BO-11X formulation (or a BO-11Xm formulation) according to theirsensitivity to immunotherapy such as with an anti-PD1, anti-PD-L1 oranti-CTLA4 antibody. These cancers include melanoma, non-small cell lungcancer, cancer of the head and neck, renal cell cancer, bladder cancer,hepatocellular carcinoma and Hodgkin's lymphoma. Alternatively, a BO-11Xformulation (or a BO-11Xm formulation) may be administered for treatingcancer types which have not previously demonstrated any sensitivity toimmunotherapy, such as prostate cancer, breast cancer, colorectal canceror subtypes of these cancers that for example have a high mutationalburden (or microsatellite instability), such as those presenting JAK,POLE, or other mutations detected by gene sequence and/or expressionprofiling affecting response to interferon, in particular interferongamma, as reported in literature (Ayers M et al. 2017; Budczies J etal., 2016; Gong J et al. 2017; Shin D S et al., 2017; Zaretsky J M etal. 2016).

In general, BO-11X formulation can be also combined with miRNAs, (orother non-coding RNA molecules that act as inhibitors or modulators ofthe expression of specific genes, such as RNAi shRNA, or siRNA; Ling H,2016; Larsson M et al., 2017), mRNA (i.e., RNA coding for proteins wheninto cells), or any other RNA-based drugs, in which a BO-11X formulationsuch as a BO-112 formulation may improve RNA delivery, activity, and/orstability (e.g. miRNAs to silence IDO, TGFbeta, or NKG2D ligand) bymeans of this combined or co-administration method. Synergisticcombinations of BO-112 formulation with complementary mechanisms ofaction would include drugs that are designed to reduce theimmunosuppression and T cell exhaustion, to potentiate T cell activationor to increase numbers of tumor specific T cells. These drugs can targettumor cells, T cells or other cells in the tumor microenvironment andinclude molecules such as immunomodulatory mAbs (CTLA4, PD1 or PDL1,LAG3, TIM3, CD137, OX40, GITR, CD40, CD25), activatory cytokines (IL2,IL12), other neutralizing mAbs (TGFbeta, IDO1, IL10, NKG2d ligand),CAR-T cells, or cancer antigen vaccines. Present data also supportcombination with agents that target Tregs and drugs involved (andtargeting) in DNA repair and/or replication.

In some embodiments, the terms “patient” and “subject” are usedinterchangeably. In some embodiments, the subject and/or animal is amammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow,pig, rabbit, sheep, or non-human primate, such as a monkey (e.g. baboon)or chimpanzee.

In some embodiments, methods of the invention are useful in treatment ahuman subject. In some embodiments, the human is a paediatric human. Inother embodiments, the human is an adult human. In other embodiments,the human is a geriatric human. In other embodiments, the human may bereferred to as a patient or a subject. In some embodiments, the human isa female. In some embodiments, the human is a male.

Treatment Regimens and Combination Therapies

In one embodiment, the present invention relates to a BO-11X composition(or a BO-11Xm composition), preferably a BO-112 composition (or aBO-112m composition), as defined herein, for use in treatment of a cellgrowth disorder characterized by abnormal growth of human or animalcells, comprising the following steps:

-   -   (i) administering to said subject (patient) according to at        least one treatment with said composition or said formulation;        followed by    -   (ii) a follow-up (analysis) of the medical conditions of said        subject (patient) and/or a follow-up of gene expression within        tumor, cancer and/or immune cells thereof; together with or        followed by    -   (iii) evaluation of (a) cell infiltrates within tumor biopsies        (e.g. CD8 and/or CD4-positive T cells), (b) necrosis and/or        apoptosis of cancer cells in tumor biopsies, or (c)        increase/decrease of circulating immune cells in blood.

This approach is useful for evaluating administration of a BO-11Xformulation such as BO-112, and can be further developed according toavailable technologies, for example in connection to imaging forevaluating tumor burden and/or response (see also Example 3).

In other embodiment, such as those summarized in FIGS. 14 and 15, thepresent invention relates to:

-   -   (i) a BO-11X composition (or a BO-11Xm composition), preferably        a BO-112 composition (ora BO-112m composition), as defined        herein, or    -   (ii) a formulation obtainable by treating a cell or tissue from        a subject (patient) ex vivo with a BO-11x composition (or a        BO-11Xm composition), preferably a BO-112 composition (or a        BO-11Xm composition), as defined herein,    -   for use in treatment of a cell growth disorder characterized by        abnormal growth of human or animal cells, wherein said        composition is administered to said subject (patient) according        to a process comprising the following steps:    -   (i) at least one treatment with said composition or said        formulation; followed by    -   (ii) a follow-up (analysis) of the medical conditions of said        subject (patient) and/or a follow-up of gene expression within        tumor, cancer and/or immune cells thereof and/or evaluation of        (ii,a) cell infiltrates within tumor biopsies (e.g. CD8 and/or        CD4-positive T cells),    -   (ii,b) necrosis and/or apoptosis of cancer cells in tumor        biopsies, or (ii,c) increase/decrease of circulating immune        cells in blood; together with or followed by    -   (iii) at least one treatment with said composition, and/or said        formulation and/or a standard of care protocol and/or an        anti-cancer drug or treatment (already approved or within a        clinical study protocol) against which the subject (patient) was        resistant, not responding or poorly responding,    -   wherein the treatment with said composition, and/or said        formulation in step (i) and step (iii) may be performed using        the same or a different dose, means of administration        (intratumoral or subcutaneous/intramuscular), or regimen. More        preferably, the standard of care treatment, drug in clinical        development, or other treatment against which the subject        (patient) was resistant, not responding or poorly responding is        radiotherapy, chemotherapy, anti-PD-1/PD-L1 therapy (or other        immune checkpoint inhibitor), cancer antigen-based vaccination        or cell-based therapy (including CAR-T therapy). Preferably, the        gene expression involves mRNA and/or protein expression of        specific sets of proteins.

In another embodiment, the present invention relates to a BO-11Xcomposition (or a BO-11Xm composition), preferably a BO-112 composition,for use in treatment of a cell growth disorder that is characterized byabnormal growth of human or animal cells, wherein said composition isadministered to a subject (patient) according to an administrationregime comprising

-   -   (I) at least one (or first) intratumoral injection of said        composition; and    -   (II) at least one (or second) subcutaneous or intramuscular        injection of said composition,    -   wherein said at least one intratumoral injection is administered        prior to said at least one subcutaneous or intramuscular        injection.

Analogously, the present invention also relates to a method of treatmentin a subject (patient) of a cell growth disorder characterized byabnormal growth of human or animal cells, wherein a BO-11X composition(or a BO-11Xm composition), preferably a BO-112 composition (or aBO-112m composition), is administered to said subject according to anadministration regime comprising:

-   -   (I) at least one (or first) intratumoral injection of said        composition; and    -   (II) at least one (or second) subcutaneous or intramuscular        injection of said composition,    -   wherein said at least one intratumoral injection is administered        prior to said at least one subcutaneous or intramuscular        injection.

Alternatively, the administration regimen for BO-11X composition (or aBO-11Xm composition) or the corresponding method of treatment in asubject (patient) of a cell growth disorder characterized by abnormalgrowth of human or animal cells may comprise:

-   -   (i) at least a first intratumoral injection in a first lesion;        and    -   (ii) at least a second intratumoral injection in the same lesion        or if no longer present, one or more additional lesions.

Preferably, in the composition for use and said method of treatment,said subsequent intratumoral injection is performed at least 24 hoursafter said at least one intratumoral injection, even more preferably atleast 48 hours after having performed two to four intratumoralinjections, still more preferably at least one week after havingperformed four intratumoral injections. Even more preferably, in thecomposition for use and said method of treatment, the administration ofsaid at least one intratumoral injection is repeated at equal ordifferent time intervals.

These administration regimens (or corresponding methods of treatment)may also involve administering a BO-11X composition (or a BO-11Xmcomposition):

-   -   (a) before or after administering a second therapeutic agent,        said second therapeutic agent being administered by intratumoral        or peritumoral injection in the same and/or other lesion(s), by        sub-cutaneous injection, or by intramuscular injection;    -   (b) after determining if the subject is resistant, insensitive,        or poorly (or not) responding to said second therapeutic agent;    -   (c) and then a said second therapeutic agent is administered to        a subject after determining if, following the administration        BO-11X composition (or a BO-11Xm composition), there is a        statistically significant increase in number of circulating        immune cells and/or change in the expression of any of the genes        of Table I (see Example 4).

With respect to the embodiments (b) and (c) above, the secondtherapeutic agent is preferably selected from anti-CTLA4, anti-PD1,anti-PDL1, CAR-T cells, cancer antigen vaccines, or agents that targetregulatory T cells, metabolic enzymes, DNA repair and/or replication, ora protein expressed by any of the genes of Table I.

Alternatively, the administration regimen for BO-11X composition (or aBO-11Xm composition) may involve the ex vivo treatment of cells withsuch a composition. In this case, the BO-11X composition (or a BO-11Xmcomposition) for use as a medicament by administering it to a subjectaccording to an administration regimen comprising:

-   -   (i) obtaining cells from the subject;    -   (ii) contacting said cells to said composition ex vivo; and    -   (iii) Administering such cells to the subject;

Preferably the treatment of a cell growth disorder characterized byabnormal growth of human or animal cells as indicated in all theprevious embodiments is a treatment of cancer. More preferably, in thecomposition for use and said method of treatment, said at least one (orany second or other additional) intratumoral, peritumoral, subcutaneousor intramuscular injection is performed at least 24 hours after said atleast one (or first) intratumoral injection, even more preferably atleast 48 hours after having performed two to four intratumoralinjections, still more preferably at least one week after havingperformed four intratumoral injections. Even more preferably, in thecomposition for use and said method of treatment, the administration ofsaid at least one intratumoral injection is repeated at equal ordifferent time intervals. Preferably, the administration of the at leastone sub-cutaneous injection is repeated at equal or different timeintervals and/or the intramuscular injection is repeated at equal ordifferent time intervals. Much more preferably, in the composition foruse and said method of treatment, the composition administered in the atleast one sub-cutaneous injection or intramuscular injection comprisesthe same or a lower amount of polyinosinic-polycytidylic acid[poly(I:C)] than in the at least one intratumoral injection.

In some embodiments, present invention provides for specific cancertreatment regimens with BO-11X formulations (or a BO-11Xm composition),and a second, immune-modulating agent (and optionally one or moreadditional therapeutic agent). For example, in some embodiments, theBO-11X formulation, e.g. BO-112, may be administered to a patient firstto normalize tumor vascularization, optionally by reducing or ablatinghypoxia. Alternatively, such first administration of the BO-11Xformulation, e.g. BO-112, may stimulate and/or increase T lymphocytes(e.g. CD4+ and CD8+ T cells) and/or NK cells tumor and/or inhibit and/ordecrease recruitment of immunosuppressive cells (e.g. myeloid-derivedsuppressor cells (MDSCs), regulatory T cells (Tregs); tumor associatedneutrophils (TANs), M2 macrophages, and tumor associated macrophages(TAMs) to the tumor. In some embodiments, the present therapies mayalter the ratio of M1 versus M2 macrophages in the tumor site to favorM1 macrophages. the BO-11X formulations, in some embodiments, may inducea long lasting (i.e. greater than transient) vascular normalization. Forexample, BO-11X formulation-vascular normalization may last greater than1, or 2, or 3, or 4, or 5, or, or 6, or 7, or 14 days, or 21 days.Accordingly, in some embodiments, this long-lasting BO-11Xformulation-vascular normalization (or, in general, the changes inimmune cells that infiltrate tumor or circulating in blood stream) mayallow for a sustainable permissive tumor microenvironment that is morelikely to be responsive to one or more immune-modulating agents. Thatis, in some embodiments, the BO-11X formulation may potentiate, rescue,or sensitize towards immune-modulating therapy.

Alternatively, the BO-11X formulations (or BO-11Xm compositions, e.g.BO-112 or BO-112m), is administered to a patient before or afterstarting treatment with one or more immune-modulating agents. Forinstance, in some embodiments, the immune-modulatory agent targets oneor more co-inhibitory molecules and reduces or eliminatesimmunosuppression. In this favourable context, i.e. upon removal ofsuppression, the BO-11X formulations (or BO-11Xm compositions, e.g.BO-112 or BO-112m), is administered is administered to stimulate theimmune system. Alternatively, the immune-modulatory agent targets one ormore co-stimulatory molecules first and the BO-11X formulation (or aBO-11Xm composition, e.g. BO-112 or BO-112m), is administered second tobolster this effect, for example, synergistically.

Further, as described herein, the BO-11X formulation and/orimmune-modulating agent can be combined with an additional therapeuticagent in the context of, for example, co-administration, a treatmentregimen or a co-formulation.

In some embodiments, the BO-11X formulations (or a BO-11Xm compositions)and/or immune-modulating agent, optionally with an additionaltherapeutic agent, can be administered sequentially. The term“sequentially” as used herein means that the additional therapeuticagent and the BO-11X formulation and/or immune-modulating agent areadministered with a time separation of more than about 60 minutes. Forexample, the time between the sequential administration of theadditional therapeutic agent and the BO-11X formulation and/orimmune-modulating agent can be more than about 60 minutes, more thanabout 2 hours, more than about 5 hours, more than about 10 hours, morethan about 1 day, more than about 2 days, more than about 3 days, ormore than about 1 week apart. The optimal administration times maydepend on the rates of metabolism, excretion, and/or the pharmacodynamicactivity of the additional therapeutic agent and the BO-11X formulationand/or immune-modulating agent being administered. Either the additionaltherapeutic agent or the present agents may be administered first.

In some embodiments, the BO-11X formulations (or BO-11Xm compositions)and/or immune-modulating agent, optionally with an additionaltherapeutic agent, can be administered simultaneously. The term“simultaneously” as used herein, means that the additional therapeuticagent and the BO-11X formulation and/or immune-modulating agent areadministered with a time separation of no more than about 60 minutes,such as no more than about 30 minutes, no more than about 20 minutes, nomore than about 10 minutes, no more than about 5 minutes, or no morethan about 1 minute. Administration of the additional therapeutic agentand BO-11X formulation and/or immune-modulating agent can be bysimultaneous administration of a single formulation (e.g. a formulationcomprising the additional therapeutic agent and the BO-11X formulationand/or immune-modulating agent) or of separate formulations (e.g., afirst formulation including the additional therapeutic agent and asecond formulation including the BO-11X formulation and/orimmune-modulating agent).

Co-administration also does not require the additional therapeuticagents be administered to the subject by the same route ofadministration. Rather, each therapeutic agent can be administered byany appropriate route, for example, parenterally or non-parenterally.

Such a combination may lead to synergism and/or additive and/or potenteffects at a lower dose of the BO-11X formulation (or a BO-11Xmcomposition) and/or immune-modulating agent. For example, when theBO-11X formulation is combined with one or more immune-modulating agentsthe effective amount of the BO-11X formulation may be lower than what itwould be in a monotherapy. In some embodiments, the BO-11X formulationis combined with an immune-modulating agent and the effective amount ofthe BO-11X formulation is a sub-therapeutic dose, for example, when theimmune-modulating agent is combined with a BO-11X formulation theeffective amount of the immune-modulating agent may be lower than whatit would be in a monotherapy. In some embodiments, the immune-modulatingagent is combined with a BO-11X formulation and the effective amount ofthe immune-modulating agent is a sub-therapeutic dose. In someembodiments, the immune-modulating agent is combined with a BO-11Xformulation and an additional therapeutic agent and the effective amountof the additional therapeutic agent is a sub-therapeutic dose. The term“sub-therapeutic dose or amount” means that a dose or amount of apharmacologically active substance is below the dose or amount of thatsubstance that is administered, as the sole substance, to achieve atherapeutic effect. The sub-therapeutic dose of such a substance mayvary depending upon the subject and disease condition being treated, theweight and age of the subject, the severity of the disease condition,the manner of administration and the like, which can readily bedetermined by one of ordinary skill in the art. In one embodiment, thesub-therapeutic dose or amount of the chemotherapeutic agent is lessthan 90% of the approved full dose of the chemotherapeutic agent, suchas that provided in the U.S. Food & Drug Administration-approved labelinformation for the chemotherapeutic agent. In other embodiments, thesub-therapeutic dose or amount of the chemotherapeutic agent is lessthan 80%, 70%, 60%, 50%, 40%, 30%, 20% or even 10% of the approved fulldose, such as from 20% to 90%, 30% to 80%, 40% to 70% or another rangewithin the values provided herein.

In some embodiments, the effective amount of the immune-modulating agentis less than an effective amount used in monotherapy for the same cancerand/or a combination therapy with an agent besides a BO-11X formulationfor the same cancer. In some embodiments, the effective amount of theBO-11X formulation is less than an effective amount used in monotherapyfor the same cancer or clinical status, and/or a combination therapywith an agent (such as an immune-modulating agent) for the same canceror clinical status.

In some embodiments, the BO-11X formulation (or a BO-11Xm composition)is combined with one or more immune-modulating agents (e.g. 1, or 2, or3, or 4, or 5 immune-modulating agents) and, optionally, one or moreadditional therapeutic agents (e.g. 1, or 2, or 3, or 4, or 5 additionaltherapeutic agents). Such combinations may lead to synergism and/oradditive and/or potent effects at a lower dose of the BO-11X formulationand/or immune-modulating agent and/or the one or more additionaltherapeutic agents. Co-administration may be simultaneous or sequential.Further the pharmaceutical compositions including the BO-11X formulationand/or immune-modulating agent may comprise the additional therapeuticagent (e.g. via co-formulation). That is, in some embodiments, two ormore of any of the agents disclosed herein may be co-formulated.Further, in some embodiments, the BO-11X formulation and/orimmune-modulating agent may be administered to a patient that isundergoing treatment with one or more additional therapeutic agent.Further, in some embodiments, the BO-11X formulation and/orimmune-modulating agent may supplant a patient's current treatment withone or more additional therapeutic agent.

Adjuvant therapy, also called adjuvant care, is treatment that is givenin addition to the primary, main or initial treatment. By way ofnon-limiting example, adjuvant therapy may be an additional treatmentusually given after surgery where all detectable disease has beenremoved, but where there remains a statistical risk of relapse due tooccult disease. In some embodiments, the agents described herein areused as an adjuvant therapy in the treatment of a cancer. In someembodiments the therapeutic agents described herein are administered asa neo-adjuvant therapy prior to resection. In certain embodiments,neo-adjuvant therapy refers to therapy to shrink and/or downgrade thetumor prior to any surgery. In some embodiments, neo-adjuvant therapymeans a therapeutic agent described herein is administered to cancerpatients prior to surgery or other technique allowing tumor ablation.

In some embodiments the therapeutic agents described herein are usefulas a maintenance therapy after an initial treatment with a first-linetherapy, including without limitation any of the additional therapeuticagents of the present disclosure.

In some embodiments, the present invention provides a treatment regimenor a method for treating cancer or tumors in a subject that includesadministering simultaneously or sequentially a therapeutically effectiveamount of a BO-11X formulation and/or an immune-modulating agent and oneor more of the additional therapeutic agents described herein. In someembodiments, the present invention provides a treatment regimen or amethod for treating cancer or tumors in a subject that includesadministering simultaneously or sequentially a therapeutically effectiveamount of a BO-11X formulation and/or an immune-modulating agent and oneor more of the anti-cancer agents described herein, including but notlimited to chemotherapeutic agents. Suitable chemotherapeutic agents tobe used in the methods of the present invention may include thosedescribed herein. In certain embodiments, the chemotherapeutic agent isone or more of 5-fluorouracil (5-FU), doxorubicin, gemcitabine,paclitaxel, and cisplatin. By way of example, in some embodiments, thepresent invention provides combining a BO-11X formulation and/or animmune-modulating agent with one or more common cancer treatmentregimens (by way of non-limiting illustration, FOLFOX, FOLFIRI, IFL, FL(Mayo), QUASAR, Machover schedule, CAF, CMF, ECF, and FEC).

In some embodiments, the additional therapeutic agent is anantihyperproliferative agent. Antihyperproliferative agents include, butare not limited to, doxorubicin, daunorubicin, mitomycin, actinomycin D,bleomycin, cisplatin, VP16, enedyine, taxol, vincristine, vinblastine,carmustine, melphalan, cyclophsophamide, chlorambucil, busulfan,lomustine, 5-fluorouracil, gemcitabin, BCNU, or camptothecin.

In addition, the additional therapeutic agent can further include theuse of radiation. In addition, the methods of treatment can furtherinclude the use of photodynamic therapy.

Salts, Pharmaceutical Compositions and Doses

In some embodiments, the present invention provides for the formulation,compositions (where formulation and composition is used interchangeablyin the present invention), and agents described herein andpharmaceutically acceptable esters, pro-drugs, salts, solvates,enantiomers, stereoisomers, active metabolites, co-crystals, and otherphysiologically functional derivatives thereof.

In one aspect, the present invention provides agents described herein,and a pharmaceutically acceptable carrier or excipient. Thepharmaceutical composition can be in any suitable form appropriate forthe desired use and route of administration. Pharmaceutical excipientscan be liquids, such as water and oils, including those of petroleum,animal, vegetable, or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. The pharmaceutical excipients canbe, for example, saline, gum acacia, gelatin, starch paste, talc,keratin, colloidal silica, urea and the like. In one embodiment, thepharmaceutically acceptable excipients are sterile when administered toa subject. Water is a useful excipient when any agent described hereinis administered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid excipients,specifically for injectable solutions. Suitable pharmaceuticalexcipients also include starch, glucose, lactose, sucrose, gelatin,malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmono-stearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. Other examples ofsuitable pharmaceutical excipients are described in Remington'sPharmaceutical Sciences (edited by Allen, Loyd V., Jr; 22nd edition,2012).

Additionally, the pharmaceutical compositions or formulations of thepresent invention may contain adjuvants such as preservatives, wettingagents, emulsifying agents, pH buffering agents, and dispersing agents.Further, auxiliary, stabilizing, thickening, lubricating, and colouringagents can be included. Prevention of the action of microorganisms maybe ensured by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, phenol acid, and the like.The pharmaceutical compositions may also include isotonic agents such assugars, sodium chloride, and the like. Where necessary, thepharmaceutical compositions can also include a solubilizing agent. Also,the agents can be delivered with a suitable vehicle or delivery deviceas known in the art. Compositions for administration can optionallyinclude a local anaesthetic such as, for example, lidocaine to lessenpain at the site of the injection.

The pharmaceutical compositions or formulations of the present inventioncan take the form of solutions, suspensions, emulsions, drops, tablets,pills, pellets, capsules, capsules containing liquids, powders,sustained-release formulations, suppositories, emulsions, aerosols,sprays, suspensions, or any other form suitable for use. Thus, thecomposition described herein may be comprised in a capsule, tablet,pill, caplet, bottle, ampoule, sachet, syringe, cartridge, nebulizer orother container. In one embodiment, the pharmaceutical composition is inthe form of a capsule. In another embodiment, the pharmaceuticalcomposition is in the form of a tablet.

In some embodiments, the administration of any of the described agentsand compositions is any one of oral, intra-venous, and parenteral. Insome embodiments, routes of administration include, for example: oral,intra-dermal, intra-muscular, intra-peritoneal, intra-venous,sub-cutaneous, intra-nasal, epidural, sub-lingual, intra-nasal,intra-cerebral, intra-hepatic, intra-pancreatic, intravesicular,intra-vaginal, transdermal, rectally, by inhalation, or topically, forexample, to the ears, nose, eyes, or skin. In some embodiments, theadministering is effected orally or by parenteral injection. The mode ofadministration can be left to the discretion of the practitioner, anddepends in part upon the site of the medical condition and/or concurrenttreatments (being, for instance, chemotherapy, radiotherapy, or incombination with antibodies, vaccines and other cancer-targeting drugs).In some embodiments, administration results in the release of any agentdescribed herein into the bloodstream.

Any agent, pharmaceutical composition and/or formulation describedherein can be administered orally. Such agents and/or pharmaceuticalcompositions can also be administered by any other convenient route, forexample, by intravenous infusion or bolus injection, by absorptionthrough epithelial or muco-cutaneous linings (e.g., oral mucosa, rectaland intestinal mucosa, etc.) and can be administered together with anadditional therapeutic agent. Administration can be systemic or local.Various delivery systems are known, e.g., encapsulation in liposomes,microparticles, microcapsules, capsules, etc., and can be used. Inspecific embodiments, it may be desirable to administer locally to thearea in need of treatment.

In one embodiment, an agent described herein and/or pharmaceuticalcomposition and/or formulations described herein is formulated inaccordance with routine procedures as a composition adapted for oraladministration to humans. Solid dosage forms for oral administrationinclude, for example, capsules, tablets, pills, powders, and granules.In such dosage forms, the active agent is mixed with at least one inert,pharmaceutically acceptable excipient or carrier such as sodium citrate,di-calcium phosphate, etc., and/or a) fillers or extenders such asstarches, lactose, sucrose, glucose, mannitol, silicic acid,microcrystalline cellulose, and Bakers Special Sugar, etc., b) binderssuch as, for example, carboxymethylcellulose, alginates, gelatin,polyvinyl-pyrrolidone, etc., c) humectants such as glycerol, etc., d)disintegrating agents such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, sodium carbonate,cross-linked polymers such as crospovidone (cross-linkedpolyvinylpyrrolidone), croscarmellose sodium (cross-linked sodiumcarboxymethylcellulose), sodium starch glycolate, etc., e) solutionretarding agents such as paraffin, etc., f) absorption accelerators suchas quaternary ammonium agents, etc., g) wetting agents such as, forexample, cetyl alcohol and glycerol monostearate, etc., h) absorbentssuch as kaolin and bentonite clay, etc., and i) lubricants such as talc,calcium stearate, magnesium stearate, solid polyethylene glycols, sodiumlauryl sulphate, glycerylbehenate, etc., and mixtures of suchexcipients. One of skill in the art will recognize that particularexcipients may have two or more functions in the oral dosage form. Inthe case of an oral dosage form, for example, a capsule or a tablet, thedosage form may also comprise buffering agents.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirs. Inaddition to the active agents, the liquid dosage forms may contain inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ,olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Dosage forms suitable for parenteral administration (e.g.intra-venous, intra-muscular, intra-hepatic, intra-pancreatic,intra-peritoneal, sub-cutaneous and intra-articular injection andinfusion) include, for example, solutions, suspensions, dispersions,emulsions, and the like. They may also be manufactured in the form ofsterile solid compositions (e.g. lyophilized composition), which can bedissolved or suspended in sterile injectable medium immediately beforeuse. They may contain, for example, suspending or dispersing agentsknown in the art. Pharmaceutical compositions of this invention forparenteral injection comprise pharmaceutically acceptable sterileaqueous or non-aqueous solutions, dispersions, suspensions or emulsionsas well as sterile powders for reconstitution into sterile injectablesolutions or dispersions just prior to use. Examples of suitable aqueousand non-aqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol, and the like), and suitable mixtures thereof, vegetable oils(such as olive oil) and injectable organic esters such as ethyl oleate.Proper fluidity can be maintained, for example, by the use of coatingmaterials such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

Any agent described herein and/or pharmaceutical composition describedherein can be administered by controlled-release or sustained-releasemeans or by delivery devices that are known to those of ordinary skillin the art. Such dosage forms can be useful for providing controlled- orsustained-release of one or more active ingredients using, for example,hydropropyl cellulose, hydropropylmethyl cellulose,polyvinylpyrrolidone, Eudragit, other polymer matrices, gels, permeablemembranes, osmotic systems, multilayer coatings, microparticles,liposomes, microspheres, or a combination thereof to provide the desiredrelease profile in varying proportions. Suitable controlled- orsustained-release formulations can be readily selected for use with theactive ingredients of the agents described herein. The invention thusprovides single unit dosage forms suitable for oral administration suchas, but not limited to, tablets, capsules, gelcaps, and caplets that areadapted for controlled- or sustained-release.

Formulations comprising the agents described herein and/orpharmaceutical compositions of the present invention may conveniently bepresented in unit dosage forms and may be prepared by any of the methodsknown in the art of pharmacy. Such methods generally include the step ofbringing the therapeutic agents into association with a carrier, whichconstitutes one or more accessory ingredients. Typically, theformulations are prepared by uniformly and intimately bringing thetherapeutic agent into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct into dosage forms of the desired formulation (e.g. wet or drygranulation, powder blends, etc., followed by tableting usingconventional methods known in the art).

It will be appreciated that the actual dose of the agents describedherein and/or pharmaceutical compositions of the present invention to beadministered according to the present invention may vary according tothe particular agent, the particular dosage form, and the mode ofadministration. Many factors that may modify the action of the BO-11Xformulations (e.g. body weight, gender, diet, time of administration,route of administration, rate of excretion, condition of the subject,drug combinations, genetic disposition and reaction sensitivities) canbe taken into account by those skilled in the art. Administration can becarried out continuously or in one or more discrete doses within themaximum tolerated dose. Optimal administration rates for a given set ofconditions can be ascertained by those skilled in the art usingconventional dosage administration tests.

Individual doses of the agents described herein and/or pharmaceuticalcompositions of the present invention can be administered in unit dosageforms (e.g., tablets or capsules) containing, for example, from about0.01 mg to about 1,000 mg of poly(I:C) molecules within the BO-11Xformulation or BO-11Xm formulation, e.g. wherein said individual BO-11Xformulation is formed by making a complex from about 0.01 mg to about 10mg of poly(I:C)] preferably about 0.5 mg to about 2.5 mg of poly(I:C),more preferably about 1 mg to about 2 mg of poly(I:C), inclusive of allvalues and ranges there between. In some embodiments, the agentsdescribed herein and/or pharmaceutical compositions of the presentinvention are administered at an amount of from about 0.01 mg to about1000 mg of poly(I:C) molecules within the BO-11X formulation daily, orfrom about 0.1 mg to about 10 mg daily [e.g. wherein said individualdaily BO-11X formulation is formed by making a complex from about 0.01mg to about 1000 mg, preferably about 0.01 mg to 10 mg of poly(I:C)],inclusive of all values and ranges there between.

In some embodiments, a suitable dosage of the agents and/orpharmaceutical compositions of the present invention is in a range ofabout 0.001 to about 10 mg of poly(I:C) molecules within the BO-11Xformulation/kg of body weight of the subject, preferably 0.003 to about10 mg of poly(I:C) molecules within the BO-11X formulation or BO-11Xmformulation (as calculated with respect to a subject or, if needed, ascalculated with respect to the body weight of the subject), morepreferably 0.005 to about 10 mg of poly(I:C) molecules within the BO-11Xformulation/kg of body weight of the subject and even more preferably0.01 to about 10 mg of poly(I:C) molecules within the BO-11X formulationper kg of body weight of the subject [e.g. wherein said individualBO-11X formulation administered is formed by making a complex from about0.005 mg to about 10 mg of poly(I:C)] per kg of body weight of thesubject, preferably about 0.003 mg to about 10 mg of poly(I:C)] per kgof body weight of the subject, more preferably about 0.001 mg to about10 mg of poly(I:C)] per kg of body weight of the subject, inclusive ofall values and ranges there between 0.01 mg/kg to about 10 mg/kg of bodyweight of the subject, inclusive of all values and ranges there between.In other embodiments, a suitable dosage of the BO-11X formulation and/orimmune-modulating agent and/or additional therapeutic agent is in arange of about 0.01 mg/kg to about 10 mg/kg of body weight, or in arange of about 0.05 mg/kg to about 1 mg/kg of body weight.

In accordance with certain embodiments of the invention, the agentsand/or pharmaceutical compositions described herein may be administered,for example, more than once daily, about once per day, about every otherday, about every third day, about once a week, about once every twoweeks, about once every month, about once every two months, about onceevery three months, about once every six months, or about once everyyear. In one embodiment a suitable dosage of the agents and/orpharmaceutical compositions of the present invention is in a range of0.1 to 10 mg of poly(I:C) molecules within the BO-11X formulation persubject (patient), preferably 0.5 to 2 mg of poly(I:C) molecules withinthe BO-11X formulation per subject, more preferably 0.6 to 1 mg persubject over an cycle of treatments. In particular, when a compositionof the present invention is administered. by intra-cutaneous orintratumoral injection, the total amount of composition can be adaptedconsequently.

Kits

The invention also provides kits that can simplify the administration ofthe agents and/or pharmaceutical compositions described herein. The kitis an assemblage of materials or components, including at least one ofthe agents described herein. The exact nature of the componentsconfigured in the kit depends on its intended purpose. In oneembodiment, the kit is configured for the purpose of treating humansubjects.

Instructions for use may be included in the kit. Instructions for usetypically include a tangible expression describing the technique to beemployed in using the components of the kit to affect a desired outcome,such as to treat or prevent, for example, cancer, gynaecologicaldisorders, or infections. Optionally, the kit also contains other usefulcomponents, such as, diluents, buffers, pharmaceutically acceptablecarriers, syringes, catheters, applicators, filters, (micro)needles,pipetting or measuring tools, bandaging materials or other usefulparaphernalia as may be readily recognized by those of skill in the art.In particular, the kit may include a device for more precise (egimage-guided) intratumoral or peritumoral drug delivery, implantable (ornot) drug eluting devices in the format of matrices (including porous,biodegradable, and/or polymeric structures and scaffolds), encapsulationsystem, or other appropriate packaging for clinical use.

Since the BO-11X compositions or BO-11Xm compositions can be useddirectly, in combination with other compounds (e.g. vaccines, drugs,adjuvants, etc.), using different routes of administration, and/orinvolving the retrieval and analysis of cells or samples from thepatients, the kit may include specific materials such as syringes forintra-tumoral, intra-intra-muscolar, and/or sub-cutaneous injections(pre-filled or not), vehicle for dilution, vials for storing biologicalmaterials from the patients, and/or antibodies for detecting specificcell markers.

EXAMPLES Example 1: Structural Characterization of jetPEI-basedpoly(I:C) Preparations (BO-11X Formulations) and Initial, In VitroFunctional Validation

Materials & Methods

Poly(I:C) Preparation

The single-stranded polyinosinic acid [poly(I)] and polycytidylic acid[polyl] molecules used for generating double-strandedpolyinosinic-polycytidylic acid [poly(I:C)] molecules] were obtainedfrom commercial providers such as Tide Group, Carbogen, Hangzhou FstPharmaceutical Co, or Invivogen. Depending from the provider and thebatch, the size distribution for pI(C) molecules is defined as being:<400 bases, 20-82% (with further tests performed using preparationspresenting, for instance, 33%, 43%, 50%, 70%, or 73% s); 400-850 base,15˜40% (with further tests performed using preparations presenting, forinstance, 20%, 27%, 30%, 34%, or 37%); 850-5000 bases, 3˜50% (withfurther tests performed using preparations presenting, for instance, 5%,6%, 13%, 30% 34%, 42%, or 48%); >5000 bases, 1% or less (generallyabsent). Depending from the provider and the batch, the sizedistribution for poly(I) molecules is defined as being: <400 bases,80-99% (with further tests performed using preparations presenting, forinstance, 81%, 86%, 91%, 95%, or 98%); 400-850 bases, 1-20% (withfurther tests performed using preparations presenting, for instance, 6%,8%, 12%, or 17%); 850-5000 bases, 0˜5% (with further tests performedusing preparations presenting, for instance, 3%, 1% or below); >5000bases, 1% or less (generally absent). Acceptance criteria formanufacturing BO-11X formulations that apply to poly(I) anloly(C) powderor solutions also include maximum absorption (at wavelength of 248±1 nmand of 268±1 nm for poly(I)Id poly(C), respectively), endotoxin content(≤10 EU/m-), pH (6.0-8.0), and sedimentation coefficient (≥4S).

Batch poly(I) preparatil and poly(C) preparations were obtained,annealed, and purified as described in PCT/EP2016/078078. Briefly,poly(I) preparlons and poly(C) preparations were obtained separatelylingpowder poly(C) in the same manner and at the same concentration.Additional steps of filtration could be implemented to further improvequality of starting solutions using membranes with a 300 kDa cut-off ora 500 kDa cut-off (Pellicon 2 cassette, Millipore). The permeates ofthese filtration steps are concentrated and freed from small sizeimpurities, such as monomers, over a 30 kDa membrane (Pellicon 2cassette, Millipore). The resulting retentate for each solution is mixedwith a concentrated buffer solution (such as PBS 10×). For bothsolutions, the optical density was determined to calculate theconcentration as a basis for a 1:1 stoichiometry for the followingannealing step, adjusting consequently the total volume before annealingstep. Poly(I) solution is mixed and stirred with poly(C) solutionmolecules at 55-62° C. for 30 minutes. The resulting solution is slowlycooled down at room temperature for approx. 3 hours for annealingsingle-stranded molecules and generating poly(I:C) molecules, andfinally filtered over a G3 glass pore filter (pore size of approx. 15-40μm).

This annealing process generates a solution containing a pool ofdifferent double-stranded poly(I:C) molecules that is then applied on achromatographic GPC column. The chromatography is performed with Omnifitglass column of 5 cm diameter that was filled with a slurry of 700mLToyopearl HW-65F in 40 mM sodium phosphate buffer. The slurry wasallowed to settle slowly, followed by washing with 40 mM sodiumphosphate buffer (pH=6.9) at increasing flow rate from 10 mL/min to 60mL/min. The column was installed in a preparative HPLC device consistingof two feed pumps, a UV detector, sampling valves and a computer. Thereaction mixture from the annealing was loaded on the column and elutedwith 40 mM sodium phosphate buffer (flow=50 mL/min, pH=6.9). Targetfractions were taken when the UV signal was between 100 mV and 1250 mV,and pooled for further work-up using four desalting cycles of dilutionand concentration using a tangential flow device (TFF, MilliporePellicon 2, regenerated cellulose, equipped with three membranecassettes of 0.1 m² each, 300 kDa cut-off). Inlet and outlet wereconnected to the first glass bottle with the pooled chromatographyfractions. Final retentate and washing solution were filtered over amembrane to give a clear, colorless solution that was desalted usingisopropanol, freeze-dried, and lyophilized at room temperature (at 1mbar for approx. 5 days). A preparation of poly(I:C) can be obtained bythe following exemplary process: poly(C) solution was heated at 61 to66° C. for 1.5 h before mixing this with the poly(I) solution andstirring at 55 to 58° C. for 70 minutes, after which the mixture wascooled and filtered over a 0.2 μm membrane.

Different commercial In vivo-JetPEI [having an average molecular weightcomprised between 8.3 and 22.5 kDa, and a polydispersity index <1.5, asdetermined from that of PEOX (polyethyleneoxide, precursor to PEI) byGel Permeation Chromatography (GPC: SOP GPC-0044) and sterile filteredthrough a 0.2 μm filter] was obtained from PolyPlus (catalog no.201-50G).

Some conditions applicable to the chromatography and/or filtration step,absence or presence of a freezing step, together with buffer andannealing time, were adapted for further reducing solution viscosity orprecipitation of complexes. Either Mannitol or Glucose is used asexcipient in the final formulation. Solution 1 containing JetPEI isobtained by either using JetPEI in a concentrated liquid preparation orsolubilizing solid bulk preparations of JetPEI (having a molecularweight comprised between 17 and 23 kDa) in an amount of sterile waterfor injection to reach 150 mM, and mixing for obtaining a homogeneoussolution. A further dilution step is performed to reach a concentrationof 11.25-11.7 mM, before the final dilution to 5.62-5.85 mM in the finalvial. Solution 2 contains poly(I:C) molecules and glucose monohydrate inan amount that, after mixing with JetPEI, provides a solution containing5% glucose (weight/total volume of said composition) and poly(I:C) at0.5-0.7 mg/mL of the total volume of said composition, whereby saidpoly(I:C) complexes with said JetPEI.

Solution 1 and 2 are independently sterilized using a double filtrationthrough 0.2 μm filters (Sartopore® 2 150 0.2 μm, fully validated assterilizing grade filters (according to ASTM F-838-05 guidelines) usinga pump Watson Marlon (speed 30 rpm). The automated mixing of the twosolutions is performed in each vial using a sequential process: (i)Solution 1 is added to the vial using a Watson-Marlow pump to dose5.95-6.05 g (6 mL; density: 1 g/mL), (ii) Solution 2 is added over thesolution 1 using a 1.8 mm internal diameter tube connected to a G20-0.9μm needle using Flexicon pump at 550 rpm speed to dose 6.08-6.40 g (6mL). Results can be improved by using a T-piece mixer. In case ofaggregates of particles (e.g. with a size in the range of 1-100 μm orlarger) that may be still present by visual inspection at the end of themanufacturing process (or during its storage) due to electrostaticinteractions, the product can be filtered over a 0.8 μm filter prior touse (for instance, before its injection), without altering neitherbiological properties nor mono-modal diameter distribution of theparticles within the composition. For example, the BO-112 formulationcan be filtered through a Minisart Syringe Filter (Sartorious) with anexclusion size of 0.8 μm. Vials (having variable volumes of BO-112formulations, e.g. 1 mL, 2 mL, 5 mL, 10 mL, or more) are sealed withsterile pyrogen-free rubber stoppers and crimp with aluminum capsulesand individually labelled. These vials can be used directly forinjections or their content can be diluted in an appropriate vehicleprior to the use.

The size of poly(I:C) molecules within BO-11X preparations wasdetermined and compared by chromatography or by using agarose gels andunlabeled or [32P] labeled poly(I) and poly(I:C) preparations. Briefly,1 μg of poly(I) and poly(I:C) (PBS) are loaded into the agarose gel andelectrophoresis was performed for 1 hour at 80 volts in TBE buffer.Depending from the size distribution of initial poly(C) and poly (I)molecules, the size distribution of poly(I:C) molecules that are presentin BO-11X preparations was determined also by chromatography as being:<400 bases, 7-57% (with further tests performed using preparationspresenting values comprised between 10% and 30%, for instance, 11%, 15%,17%, 21%, 26%, or 28%); 400-850 bases, 20-45% (with further testsperformed using preparations presenting values comprised between 20% and30%, for instance, 23%, 25%, or 27%); 850-5000 bases, 20-70% (withfurther tests performed using preparations presenting values comprisedbetween 40% and 60%, for instance, 42%, 45%, 52%, 53%, or 55%); >5000bases, 0-9% (with further tests performed using preparations presentingvalues comprised between 0% and 5%, for instance, 3%, 1%, or 0%). Thesize of different batch poly(I) preparations and poly(C) preparationswere evaluated and compared by chromatography.

BO-112 compositions are produced and validated at a concentration andwith particle size distribution as described for BO-11X formulations inPCT/EP2016/078078. BO-112 compositions are provided as vials containingpoly(I:C) at 0.5-0.8 mg/mL concentration. The stability of BO-112compositions in these vials was confirmed after 1 year of storage at2-8° C.

Commercially Available Poly(I:C)-Containing Formulations

Poly-ICLC is a poly(I:C) preparation that is stabilized with polylysineand carboxymethyl-cellulose (Ewel C et al., 1992; WO2005102278).LyoVec-HMW (Cat. No. tlrl-piclv) and LyoVec-LMW (Cat. No. tlrl-picwlv),and corresponding poly(I:C) preparations having high molecular weight(HMW; Cat. Name tlrl-pic) and low molecular weight (LMW; Cat. Nametlrl-picw) are available from Invivogen.

Analytical Technologies

The value for zeta average (z-average) diameter and polydispersity indexof JetPEI/poly(I:C) particles in distinct BO-11X preparations (between0.5-0.8 mg/mL, to be diluted for cell-based and other assays at apoly(I:C) concentration of 1.0 μg/mL) were determined using ZetasizerNano ZS according to the manufacturer's instructions and in accordancewith ISO 22412, based on the assumption that said particles arespherical. In general, dynamic light scattering (Nanosizer technology)is applied using v7.11 software.

In Vitro Characterization of BO-11X Preparations

The different poly(I:C)-based preparations were tested using humanmelanoma cells, human pancreatic cells, or human melanocytes accordingto the literature describing the properties of BO-110 complexes on humanmelanoma cell line SK-MEL-103 and human pancreatic cancer cell line c(Pozuelo-Rubio M et al., 2014; Tormo D et al., 2009; WO2011003883).Briefly, cell viability assays were performed on adherent cells at least12 hours before treatment. The percentage of cell death at the indicatedtimes and treatment concentrations was estimated by standard trypan blueexclusion assays on floating and adherent cells that were pooled,stained with a 0.4% trypan blue solution (Gibco Laboratories, GrandIsland, N.Y., USA) and scored under a light microscope (a minimum of100-500 cells per treatment were counted). Each preparation was testedfor a period comprised between 12 hours and 48 hours and atconcentrations poly(I:C) molecules in the different preparations thatwere comprised between in a range between 0.3 and 2.5 μg/ml.

The death-inducing activity of BO-112 was tested in normal melanocytesand cell lines from melanoma and glioblastoma and compared to isolatedcomponents, i.e. poly(I:C) molecules and linear PEI (Jet PEI; Polyplus).Normal melanocytes were isolated from foreskins of asymptomatic donors.Melanoma cells SK-Mel-28, SK-Mel-103, and UACC62 (with mutations in p53,NRAS and BRAF respectively) were obtained from established collectionsat the ATCC or Memorial Sloan Kettering Cancer Centre (USA) and weresubject to short tandem repeat (STR) profiling (GenePrint® 10 System)for cell line authentication. Primary cells from melanoma patients wereobtained and maintained using standard protocols. Cells were plated on96 well plates (6000 cells/well). In triplicate per experiment, withPoly(I:C) only, BO-110 or BO-112 formulations at 0.5 or 1 μg/ml for a 24hrs or 40 hrs treatment.

Tumor Cell Death Characterization

Mouse cancer cell line B16-F10, skin melanoma, (ATCC code CRL-6475) wasobtained from established collections at the ATCC; B16-OVA cells(derived from B16-F10 cells) were obtained from the Centro deInvestigación Médica Aplicada (Navarra, Spain). B16 cell lines that areinsensitive to IFNs were obtained from Invivogen (B16-BIue™ IFN-γ cells,Cat.no. bb-ifng; B16-BIue™ IFNα/β cells, Cat. no. bb-ifnt1) andmaintained as indicated by manufacturer. The characterization of thetumor cell death (apoptosis, necrosis, immunogenic cell death) inducedby BO-112 formulation was investigated and compared with isolatedpoly(I:C) and with other TLR ligand (LPS, specific for TLR4). B16-OVAcells (10⁵ cells/well) were cultured: alone, with ultrapure LPS (0.25and 1 μg/mLl, InvivoGen, San Diego, Calif.), with BO-112 formulation(0.25, 0.5 and 1 μg/ml), or with poly I:C only (0.25, 0.5 and 1 μg/ml)for 48 hours. Cell apoptosis and necrosis rate were analysed by flowcytometry with Annexin V and 7ADD combined staining (Living cells: 7ADDnegative, Annexin V negative-; cell necrosis: 7ADD positive, Annexin Vnegative; early apoptosis 7ADD negative, Annexin V positive; lateapoptosis: 7ADD positive, Annexin V positive). The effect of BO-112 oninducing immunogenic cell death was analysed by flow cytometry bydetecting the cell surface expression of MHC-1, CD95 and Calreticulin.For flow cytometry, samples were acquired in a Gallios Cytometer(Beckman Coulter) and data analyzed with Kaluza Flow Analysis Software(Beckman Coulter).

PARP Activation

In addition, the effect of BO-112 formulation on the activation of theenzyme Poly ADP ribose polymerase (PARP), which is involved in DNArepair, genomic stability, and programmed cell death, was investigated.MC38 colon adenocarcinoma (Cellosaurus code CVCL_B288, Kerafast cat. no.ENH204), and 4T1 breast cancer cells (ATCC code CRL-2539) aere obtainedfrom established cell collections such as the ATCC. Each cancer cellline (3×10⁶) wasstimulated with BO-112 (0.5 μg/mL) and PARP activationwas analysed at 0, 16 h and 24 h in the cell lysates by WesternImmunoblotting. PARP Monoclonal Antibody (clone C-2-10, ThermoFisherScientific) was used to identify a 116 kDa protein which corresponds toPARP and the 85 kDa apoptosis-induced cleavage product.

Results

PCT/EP2016/078078 discloses protocols and experimental data about theinitial development and characterization of BO-11X formulations, leadingto increased cytotoxicity of BO-11X formulations against cancer cellswhen compared to BO-110 complexes as described in the literature andobtained on a laboratory scale (Pozuelo-Rubio M et al., 2014; Tormo D etal., 2009; WO2011003883). The data on the BO-11X manufacturing processand resulting poly(I:C)-containing preparations in GMP conditions withthe processes and preparations were compared to those disclosed in theliterature.

FIG. 1A provides an overview of such process for generating a first typeof BO-11X preparations that are named BO-112 formulations wherein thedrug substance (i.e. double stranded poly(I:C) molecules that aregeneratedl annealing of poly(I) and poly(C) single-stranded molecules)is first mixed with an excipient like glucose in a solution that issterilized by filtration, separately from the solution containing apolymer having the function of carrier (i.e. JetPEI). Then, these twobulk preparations are appropriately mixed into each vial to generate alarge number of structurally and functionally comparable pharmaceuticalformulations that are required for pharmaco-toxicological studies andclinical applications. This process may also provide poly(I:C)formulations in which a further compound (such as an immune-relatedadjuvant or other therapeutic compound) is present in the particles (andthen in the final formulation) that are formed in Stage 1, in particularby adding such therapeutically relevant compound together with (or as analternative to) an excipient like glucose, providing an example ofBO-11Xm formulation that can be named BO-112m.

At least some of such reproducibility and acceptance criteria can becompared to the ones of other poly(I:C)-containing formulations forwhich anticancer activity are known. At the level of poly(I:C) moleculessize, the poly(I:C) molecules that are included in the commercialLyovec-HMW and Lyovec-LMW are covering size ranges that are clearlydistinct from those provided by the BO-11X manufacturing process (withHMW almost entirely above 0.85 kb and LMW almost entirely below 0.85kb). This size difference in poly(I:C) molecules may be dependent fromthe different manufacturing process and/or carrier that are associatedin the complexes with poly(I:C) molecules. The Z-average values ofcomplexes within poly(I:C)-based complexes within poly-ICLC (comprisingpolylysine and carboxymethylcellulose) and LyoVec-HMW/LyoVec-LMW(according to the manufacturer, comprising the cationic lipid-basedtransfection reagentsDi-tetradecylphoshoryl-N,N,N-trimethylmethanaminium chloride, or DTCPTAand the neutral lipid 1,2-Diphytanoyl-sn-Glycero-3-Phosphoethanolamine,or DiPPE) were compared to the one of BO-112 formulations, showing thatthese commercial formulations contain complexes that are much bigger (inlarge majority larger than 200 nm) and, at least for Lyovec-LMW, with abi-modal distribution (FIG. 1B). If this analysis is performed after afreeze/thaw cycle, these commercial preparations appear also as lessstable, with a variability not observed for BO-112. Indeed, if BO-112formulation a Z-average diameter (d. nm) of 100+/−50 nm (e.g. 82.5 nm),and without exceeding 400 nm, LyoVec-based and Poly-ICLC formulationshaving a Z-average value well above 300 nm, thus confirming thatcommercially available poly(I:C) are provided as preparations that areeither heterogeneous in composition or include large particles that arepoorly characterized functionally and whose size is modified during afreeze/thaw cycle.

This approach can be automated for providing BO-11X formulations (suchas BO-112 preparations) with even more uniform features. Thismanufacturing procedure allows not only to preventing having freeJetPEI, or poly(I:C) molecules in solution and not complexed withinBO-112 preparations but also obtaining a controlled average diameter andthe mono-modal diameter distribution of the complexes within BO-112preparations, so that the Z-average can be modulated between 30 and 150nm. Moreover, this approach can be adapted to include poly(I:C)molecules of different size distribution, as well as otherpolyribonucleotides (Poly (A), Poly(G),and/or Poly (U)) having differentsize, distribution of size, and/or presenting single and double-strandedregions.

PCT/EP2016/078078 also discloses that commercially available poly(I:C)are provided as preparations that are either heterogeneous incomposition or include large particles that are poorly characterizedfunctionally and whose size is modified during a freeze/thaw cycle.Indeed, hyperchromicity can be also used to evaluate BO-112 formulation,and in particular the stability of double-stranded poly(I:C) moleculeswithin the particles as a consequence of changes in temperature (orother condition) determining the separation between poly(I) strands andpoly(C) strands. BO-112 formulation shown a very low hyperchromaticeffect with differences in transmittance at 260 nm lower than 0.2 or0.1. Stability of frozen BO-11X vials at −20° C. for different time hasbeen also assessed and confirmed, at least up to one month.

Filtration and freezing of BO-112 formulation prior to administrationdoes not promote substantial modifications to the cytotoxic propertiesor stability on the particles within the composition with respect to theoriginal BO-112 formulation. For instance, compositions maintain D90%below 250 nm, zeta potential superior to 30 mV (e.g. between 40 mV and50 mV), hydrodynamic diameter with a Z-average between 30 and 150 nm,compatibility with the use of glucose as excipient, polydispersityvalues comprised between 0.1 and 0.6, and other applicable criteria fromEuropean Pharmacopoeia).

Thus, BO-11X preparations, such as BO-112 preparations, are formulationsthat present the high level of stability and reproducibility forparticles formed by poly(I:C)-JetPEI complexes having Z-average diameter(d.nm) below 200 nm (when not below 100 nm) that are not observed forcommercial poly(I:C) formulations that are based on other carriers andmanufacturing methods. The resulting BO-112 preparations present BO-112complexes having a mono-modal diameter distribution, without visibleparticles (i.e. without a number of particles above the limit requiredby Eur. Pharm. 2.9.20), even if the final solution is not filteredthrough 5 μm after mixing the bulk solutions 1 and 2. BO-112preparations can be filtered over a 0.8 μm filter prior to use (forinstance, before its injection), therewith altering neither biologicalproperties nor mono-modal diameter distribution of the particles withinthe composition. For example, the BO-112 formulation can be filteredthrough a Minisart Syringe Filter (Sartorious) with an exclusion size of0.8 μm. The mixing conditions can be adapted, in particular by modifyingthe mixing speed between 50 rpm and 600 rpm and/or the flow speed foreither poly(I:C) or JetPEI Solution between 1 mL/min and 50 mL/min.

In general, BO-11X preparations (and in particular BO-112 preparations)present the following main features: colorless, no visible particles anosmolality comprised between 220 and 340 mOsm/kg, a pH comprised betweenpH 2 and pH 4 (e.g. between 2.7 and 3.4), an optical rotation between+1500 and +3750, a zeta potential equal or superior to 30 mV, amono-modal diameter distribution of particle with Z-average diameter(nm) between 30 and 250 nm (for instance, between 30 and 150 nm), butpreferably between 60 nm and 130 nm, and comprising poly(I:C) molecules,wherein at least 40%, 50%, or 60% of such double-strandedpolyribonucleotides having a size higher than 0.85 Kb and at least 70%of such double-stranded polyribonucleotides have a size comprisedbetween 0.4 and 5 Kb. Features such as diameter distribution of theparticles can be modified by using T-piece mixer in combination withdifferent flow speed for both Solution 1 and Solution 2. When such speedis above 20 mL/min (e.g. 30 mL/min), the turbidity of the resultingBO-112 preparation is reduced in parallel with the reduction ofZ-average particle diameter and diameter distribution around this value,while maintaining mono-modality, possibly due to the change in the flowregime.

The exemplary BO-112 preparation is provided as vials comprisingparticles having a Z-average diameter of between 45+/−5 nm and 81+/−5 nm(e.g. 73+/−5 nm), with at least 50% of particle smaller than 85+/−20 nm,polydispersity of 0.25, the zeta potential of 38 mV, and pH 3.1. Thesestructural properties that are maintained after freeze/thaw cycle at−20° C. or extensive exposure at room temperature can be modified indistinct batches, maintaining the acceptance criteria within specificranges of values. In particular, when GMP and non-GMP batches arecompared for physicochemical and functional properties and morereproducible and effective criteria for evaluating BO-112 formulation(and BO-11X formulations in general), more preferred ranges are defined.For instance, terapeuticallly and biological effective BO-112formulations can present particles having an Z-average diameter of100+/−50 nm or of 80+/−20 nm (e.g. 76 nm, 89 nm, or 96 nm), with apotential z comprised between about 35 (or 40) and 50 mV (e.g. 39 mV, 46mV, or 50 mV) or about 40 and 45 mV (e.g. 43 mV), at least 90% ofparticle has a diameter below 250 nm or of 200+/−50 nm (e.g. 170 nm, 172nm, 174 nm, 216 nm, 218 nm, 220 nm), and polydispersity between 0.2 and0.3 (e.g. 0.21, 0.23, or 0.25). Additional criteria can be associated tofeatures that are measured in the aqueous composition such as presenceof monomeric inosinic acid below detection or irrelevant (e.g. below 1,0.5, 0.2, or 0.1 μg/mL), osmolality comprised between 300 and 310mOsm/kg, ionic strength below 5 mM or, preferably, below 1 mM (e.g. 0.7,0.76, 0.78, or 0.80). These values may be modified followingpreservation at low temperatures (e.g. 5° C.+/−3° C.) but they shouldstill remain within these ranges.

The Poly(I:C) and particle concentration in the vial can be adaptedaccording to the final use. However, in order to maintain the stabilityof Poly(I:C)- and PEI-based complexes in the compositions, the vialshould comprise BO-11X with a concentration superior to 0.1 mg/mL ofPoly(I:C), possibly superior to 0.5 mg/mL of Poly(I:C) molecules, sothat zeta-potential and aggregation features are stable and allow auniform and effective use of the BO-11X preparation. The vials can beprepared for single use (containing a volume of 4 mL, 2 mL, 1 mL ofBO-11X composition or less) as in larger batch preparations (containing5 mL, 10 mL, 15 mL or more of BO-11X composition or more) that can befractionated in 2, 3, 5 or more aliquots for single use that areadministered over the period of treatment (3 days, 5 days, 7 days, 10,days, 15 days, 30 days or more).

If the cytotoxic activity of BO-112 formulation is compared withcommercial formulations, the latter ones appear much less effective inkilling cancer cells in at least two in vitro models (FIGS. 2A and B).The cytotoxic activity of BO-11X preparations can be measured andvalidated for further uses in different types of cancer cell lines,representative of different cancer indications. These effects may bealso studied by measuring the expression and/or secretion of proteinsthat are known to modify, and possibly improve, the cellular responseagainst cancer cells. For instance, a BO-112 formulation induces, muchmore efficiently that Poly-ICLC, Interferon-beta expression in amelanoma cell line over a period of at least 24 hours (FIG. 2C). This invitro evidence can be used for evaluating not only which types of cancercan be more efficiently treated by administering a BO-11X formulationbut also for evaluating which other cancer treatments (such ascell-based or antigen-based vaccines, adjuvants, antibodies,chemotherapeutic drugs, radiotherapy, cell-based therapies,immunotherapy, epigenetic therapy, or inhibitors of enzymatic activitiessuch as kinases or metabolic enzymes) may act in a more effective mannerwhen administered in combination with a BO-11X formulation (e.g. byreducing the dosage, the frequency, and/or the period of treatment withthis other approach).

Indeed, the specificity of such cytotoxic effects against cancer celllines, and not against normal primary cells, by BO-11X formulations (aspreviously described for lab-scale BO-110 formulations; Tormo D et al.,2009) was confirmed in vitro by comparing the activities withappropriate compounds and cell controls (FIG. 3). Neither linear PEI_(L)nor poly(I:C) molecules alone affect in a significant manner theviability of tumor cells (melanoma or glioma. Only when linear PEI andpoly(I:C) molecules are complexed as provided in BO-112 formulation, asignificant killing of tumor cells, without affecting viability ofnormal melanocytes, is observed. Cell-based assays for in vitrocytotoxicity may be part of the validation and ranking of batches ofBO-112 formulation prior to further (pre-)clinical uses. Such assays canbe based on the effect on human melanoma cell lines (such as SK-Mel-19,SK-Mel-28, SK-Mel-103 and SK-Mel-147 cells) after 24, 36, or 48 hourexposure to a batch of BO-112 formulation at 1, 2 or more standardconcentrations (e.g. 0.35, 0.5, 0.85, 1.0, or 1.5 μg/mL, with or withoutprior filtration using a 0.8 μm filter) in culture medium, using vehicleor other particle-free solution having comparable criteria (such asosmolality, pH, or ionic strength) as negative control and SDS or otherbiological or chemical cytotoxic agent.

As a whole, the poly(I:C)-based, particle-based, composition-based, andcytotoxicity-based criteria that are applied for producing andvalidating BO-11X formulations (and in particular BO-112 formulations)make this formulation of poly(I:C) a structurally different andfunctionally improved formulation with respect to previouspoly(I:C)-based formulation either commercially available (see figures)and/or described in the literature (Schaffert D et al., 2011;WO2011003883; WO2015173824). With respect to these latter publications,these non-GMP, poly(I:C)-based formulations require the addition ofgroups such as PEG (with shielding activity) and/or EGF peptides (forcell targeting) in order to achieve the desired level of cytotoxicityusing particles-containing solutions having substantially higher size,at an higher N/P ratio, with additional chemical complexity inproduction, and with a specificity for cancer cells limited to thoseexpressing a cell surface antigen such as EGFR.

The observations described above were confirmed in a cell lins from amelanoma patient that have been exposed to BO-112 composition (whencompared to other poly(I:C)-based formulations) not only specificallyand more strongly reduced the viability of cancer cells but also inducedInterferon alpha/beta production by such cells (FIG. 4). Moreover, theBO-112 composition promotes tumor cell death (apoptosis and necrosis)and enhances immunogenic cell death (ICD) to a much greater extent thanLPS (a TLR4 agonist) or the corresponding poly(I:C) molecules (generallydefined as TLR3 agonist) at comparable concentration (FIGS. 5A and B).This stronger, pro-apoptotic potency of BO-112 formulation, whencompared to these TLR-specific agonists, has major implications foreliminating cancer cells but also for activating an effective anti-tumorimmune response. Indeed, a significant increase in the expression levelsof ICD markers, such as MHC-I, CD95 or Calreticulin, is observed afterBO-112 stimulation (compared to untreated cells, LPS- or polyI:C-treated cells) confirming the strong potency of BO-112 formulationto induce anti-tumor immunity. Similar in vitro studies can be performedusing tumor mouse models for in vivo studies and for guiding clinicaldevelopment.

Similar cell-based approaches have been used for validating BO-11X, andBO-112 formulation in particular, that have been exposed to filtrationand/or freezing, confirming that the cytotoxic effects are not onlyqualitatively and quantitatively maintained in BO-112 preparations aftersuch processes but also for evaluating the enhanced expression of celldeath markers and/or cancer antigens or targets specifically recognizedby immune cells or cancer drugs. For instance, this approach can be usedalso for measuring the reduced or decreased expression (or degradation)of biological targets for cancer drugs such as cell surface markers andreceptors, kinases, enzymes.

For example, aside from immune checkpoint, the activity of enzymes thatcontrol DNA repair such as Poly [ADP-ribose] polymerases may be affectedby the exposure of cells to BO-11X compositions, as it has been observedfor PARP1 in cell lines and fresh tumor specimens, derived fromautologous primary and metastatic head and neck squamous cell carcinomawhere the role of TLR3 signaling in metastatic progression has beenevaluated, observing significant apoptosis as measured by cleaved PARPin primary and metastatic tumor samples, as well as in related celllines, after the exposure to poly(I:C) molecules (Umemura N. et al.,2012). FIG. 6 shows that PARP cleavage is also observed when BO-112formulation is administered to 4T1 breast and MC38 colon cancer celllines, indicating that BO-112 formulation can be used for improving theuse of PARP inhibitors, whose clinical use Is limited by resistancemechanisms in patients (Lim J and Tan D, 2017). in combination therapiesfor expanding the therapeutic utility of PARP Inhibitors, as shown inprevious figures for anti-PD1 or antiPD-L1.

More in depth analysis of these in vitro data for guiding clinicaldevelopment can be performed using different pre-clinical modelsinvolving the production and the comparison of different BO-11Xformulations, different administration regimens, and/or conditionsassociated to a disease such as cancer. In particular, additionalstudies may involve the determination on effect of the BO-11Xformulations over the activity, expression, and/or concentration of mRNAand/or corresponding proteins in biological materials (such as blood orbiopsies) and cells obtained from patients (being cancer cells, immunecells, or other cell type). The properties of BO-11X formulations may beevaluated in human cells (being primary tumor cells, cell lines, orgenetically modified cells) when cultured in vitro, tested ex vivo,and/or when injected in mice, where more complex immunologicalmechanisms can be studied. The characterization of such activities, aswell as of associated change in gene expression and potential mechanismsof actions, may allow defining optimized administration routes andregimens, combinations with other drugs, follow-up treatments, and/orpatient populations applicable to BO-11X formulations, as summarized inExample 3.

Example 2: Functional Characterization of BO-11X Preparations in AnimalModels

Materials & Methods

BO-11X Formulations and Other Compounds

BO-112 formulations have been obtained as described in Example 1, anddiluted with a 5% glucose PBS solution (vehicle; ref: BE14-516F, Lonza,France) into three different concentrations in accordance with a dosingamount per kilo bodyweight of the animal of respectively 0.05 mg/Kg, 0.5mg/Kg and 2.5 mg/Kg.

Murine anti-PD-L1 antibody (InVivoPlus, clone 10F.9G2) was chosen ascombination immunotherapy compound. Rat-IgG2b antibody (clone LTF-2,BioXCell) was used as isotype control. Each day of injection to mice,anti-PD-L1 and Rat-IgG2b (RIgG) antibodies were diluted with vehicle atfinal concentrations of 1.5 mg/ml.

Murine anti-CD4 (clone GK1.5) and anti-CD8a (clone 2.43), and rat-IgG2b(clone LTF-2) antibodies (all from BioXCell) were used for T celldepletion. Mice received three initial doses of anti-CD4 or anti-CD8α(300 μg/mouse), or Rat-IgG2b (100 μg/dose) antibodies, followed bymaintenance doses of 100 μg/mouse.

Mouse Cell Lines

(B16-F10, skin melanoma, ATCC code CRL-6475, and B16-OVA derived fromthem; MC38, colon adenocarcinoma, Cellosaurus code CVCL_B288, Kerafastcat. no. ENH204; 4T1, breast cancer, ATCC code CRL-2539; were obtainedfrom established collections at the ATCC or from the Centro deInvestigación Medica Aplicada (Navarra, Spain) and, where possible, weresubject to short tandem repeat (STR) profiling (GenePrint® 10 System)for cell line authentication. B16 cell lines that are insensitive toIFNs were obtained from Invivogen (B16-Blue™ IFN-γ cells, Cat.no.bb-ifng; B16-BIue™ IFNα/β cells, Cat. no. bb-ifnt1) and maintained asindicated by manufacturer.

Animal Models for Human Cancers

Above-indicated cell lines were injected subcutaneously (s.c.) in theRIGHT flank (5×10⁵-1×106⁵ cells) of 8- to 10-week-old female C57BL/6mice. For abscopal studies, a second s.c. injection (3×10⁵ cells) wasperformed in the LEFT flank (directly following the primary injection oftumor cells in the right flank, concomitant injection model). Forre-challenge studies a second s.c. injection (2.5-5×10⁵ cells) wasperformed in the LEFT flank of mice that responded to BO-112 treatmentand were free of tumors. Tumors were measured by caliper weekly untilsacrificed and volume calculated (length×width2/2). Survival wasevaluated by Kaplan-Meier analysis.

BO-112 Administration and Other Compounds

BO-112 treatment started when tumor volume was 80-100 mm³. BO-112administration and vehicle was performed intratumorally via a singledirect injection into the tumor mass of the RIGHT flank. BO-112 doseschedule (2 doses/week, 3 weeks). Anti-PD-L1 and Rat-IgG2b antibodiesadministration was performed intraperitoneally, started at the seconddose of BO-11X and continued the same schedule as BO-112. Anti-CD4,anti-CD8α and Rat-IgG2b mAbs were administered intraperitoneally; aninitial dose was injected one day before starting BO-112 administration,and continued along the experiment. The exact schedule of administration(BO-112 and other compounds) of every experiment is indicated in thefigures.

Immune Response Studies

The implication of different T cell subsets in the BO-112 anti-tumoreffect was investigated by T cell depletion experiments in the MC38mouse tumor model. Anti-CD4 or anti-CD8a antibodies were administeredsystemically in MC38-tumor bearing mice starting before BO-112treatment; dosage, administration and schedule of BO-112, anti-CD4 oranti-CD8α antibodies is described previously. Tumors were measured bycaliper weekly until sacrificed and volume calculated (length×width2/2).T cell activation and priming induced by BO-112 therapy in vivo wasevaluated 24 h after the second administration of BO-112. Tumors anddraining lymph nodes were excised, tumors were digested(Collagenase/Dispase digestion medium) and filtered, all samples wereprocessed to obtain a single cell suspension. A standard protocol forflow cytometry surface and intracellular stainings was performed,fluorochrome-labeled antibodies used: anti-CD4, -CD8, -CD45, -PD-1,-IFNγ, -CD137 (Biolegend). Antigen specific CD8+ T cells against OVA orTrp2 were analyzed (iTAg Tetramer/PE-H-2 Kb, BML internationalCorporation). Samples were acquired using a Gallios Cytometer (BeckmanCoulter) and data were analyzed with Kaluza Flow Analysis Software(Beckman Coulter).

The death-inducing activity of BO-112 was tested in mouse cancer cellsthat do not respond to IFNs by an MTS assay (CellTiter 96® AQueousNon-Radioactive Cell Proliferation Assay, Promega). B16, B16-IFN-α/β (donot respond to IFN-γ) and B16-IFN-γ (do not respond to IFN-α/β) cells(5000 cells/well; 96 flat-well plates, 8 replicates per condition) werecultured with Poly(I:C) only (0.5 μg/mL), BO-112 formulation (0.5μg/mL), or 2′,3′-Cyclic GMP Sting Agonist (7 μg/mL; InvivoGen, SanDiego, Calif.) for 24 hrs, 48 hrs, 72 hrs depending on the experiment,as described in the Figures. Absorbance (OD 492 nm) was measured in anELISA reader. Dead cell % is referred to untreated cells (0%).

Results

The anti-cancer, in vivo efficacy of BO-112 formulations wasinvestigated for in vivo in an immune competent mouse strain, implantedwith mouse melanoma cells. Mice were treated either with a PBS solutionor a BO-112 formulation at three different concentrations (0.05, 0.5, or2.5 mg/kg, preferably administered intratumorally), in combination witha murine anti-PD-L1 antibody (preferably administered systemically) andcompared to vehicle alone throughout 3 weeks (FIG. 7A). Anti-PD-L1antibody in combination with the vehicle did not significantly increasesurvival when compared to vehicle alone. All three BO-112 formulationscombinations tested with anti-PD-L1 (and possibly independently formsuch antibody) significantly increased survival of the mice compared tovehicle or anti-PD-L1 alone. Moreover, survival significantly increasedin the combination of 2.5 mg/kg BO-112 formulation+anti-PD-L1 comparedto the lower doses of BO-112 formulation (FIG. 7B). Indeed, BO-112formulation administered alone clearly reduces tumor growth in thismodel (FIG. 7C-D). The effect of this combination was also tested inmodel for abscopal effects of intratumoral injections in a similarmelanoma mouse model, in which the BO-112 formulation not only reducedthe size of injected tumor mass but also the non-injected tumor mass,thus confirming both local and systemic anti-tumor effect of BO-112formulation (FIG. 8).

Anti-PD-L1 antibody is an important and validated anti-cancer drug, andmediator of an effective immune response against the cancer cells. Ourexperiment demonstrates that BO-11X complexes can be used in combinationwith other anti-cancer agents, and that the combination of BO-11Xcompounds with other anti-cancer agents such as anti-PD1 has superiorpotency to the anti-cancer agent alone, leading to significant increasesin survival and anti-tumor efficacy. Moreover, the improvement insurvival (or in immune memory cells, and more general systemictherapeutic responses) correlates with an increase in dose of BO-11Xformulation to the combination, supporting that the added benefit insurvival is mediated through the BO-11X formulation. This anti-cancer,in vivo efficacy of BO-112 formulation was confirmed, also in absence ofanti-PD-L1 or of any other treatment, in both 4T1-based (FIG. 9) andMC38-based (FIG. 10) models for breast cancer and colorectal cancers,respectively. BO-112 reduced not only tumor growth in 4T1 breastcarcinoma model but also improved mice survival in MC38 colon carcinomamodel, in the latter case also by using two different approaches (withor without a re-challenge with cancer cells; FIGS. 10 and 11).

Thus, BO-11X formulations can be used (alone or in combination withother anti-cancer agents, such as antibodies, immunotherapy, orchemotherapy, that can be administered using the same or a differentroute) in treating melanoma and other cancer indications, in particularthose allowing peritumoral or intratumoral injection such as inpancreatic, endometrial, ovarian, renal, hepatocellular or colorectalcancer. Moreover, B0112 induces systemic immunity by promoting immunecell memory allowing its use as single therapeutic agent.

At this scope, the identification of specific biological pathways andmechanisms of action may guide the most appropriate dosages, regimens,combination with other drugs or therapies, and indications for BO-11Xformulations, as shown for combined effects of immunomodulatorymonoclonal antibodies targeting PD-1, CTLA4, PD-L1, or CD137 andpoly(I:C) preparations that enhance the activities of dendritic cells(Sanchez-Paulete A R et al., 2015). To this end, Duewell P et al., 2015discloses an alternative animal model of disease that may be used fortesting the composition of the present invention for the immunotherapyof pancreatic carcinoma.

The therapeutic effect on tumor growth (locally and/or in distallocations) and the anti-tumor immune response of BO-11X formulations canbe measured by performing In vivo studies on Intratumoral (i.t.)administration across at a range of concentration for poly(I:C)molecules (such as 0.5, 1, 2, 2.5, or 5 mg/kg) to evaluate how suchtreatment improves mouse survival in a relevant model (such as a mousemelanoma, breast cancer, ovarian cancer, leiomyosarcoma, endometrialcancer, or pancreatic cancer models), with or without co-administering afurther drug or a vaccine. At the same time, dose-response studies aboutspecific biological activities induced by BO-11X treatment can beevaluated in parallel ex vivo, using human or animal samples at thelevel of apoptosis induction (by Caspase-related Glow), immunogenic celldeath induction (for example MHC-I, calreticulin, CD95, or HMGB1expression), chemokine/cytokine secretion in biological fluid (forexample, secretion of IL-6 and IP-10), in vitro cellular activationand/or proliferation (for example, associated to CD40, CD86, CD69upregulation on relevant cell types). These studies can be performedusing cells that are directly involved in disease (e.g. tumor cell,epithelial cell, endothelial or epithelial cells) or cells indirectlyinvolved since performing some immune or immunoregulatory activities(e.g. human peripheral blood mononuclear cells, NK cells, B cells,CD4+/CD8+ T cells, dendritic cells) either within tumors, in lymphoidorgans or in blood. These studies may also involve the effect of BO-11Xtreatment using different routes of administration, such assub-cutaneous or intra-muscular administration, in order to establishdifferent schedules for BO-11X clinical uses, as well BO-11X suitabilityas adjuvant in cancer-related and/or infection-related vaccinations.

These studies can be further associated to the identification tomolecules that may be used as biomarkers to predict response to BO-11X(or lack thereof) in order to stratify disease stages and/or patients'populations for BO-11X treatment. In particular, the analysis of BO-11Xactivities may be performed by comparing the qualitative and/orquantitative feature of immune cells (such as NK cells, B cells,CD4+/CD8+ T cells) and the tumor growth kinetics in animal models wherechanges in specific cell markers and (sub-)populations can be measuredalso when specific cell types are either depleted or activated incombination with BO-11X administration (for example, by co-administeringT cell depleting or activating antibodies, viral, bacterial or cancerantigens, cell-based treatments such as adoptive T-cell therapies, orcompounds with adjuvant properties).

The involvement of specific immune cells can be explored in canceranimal models similar to those previously described, where the treatmentwith BO-112 formulation is evaluated with respect to the depletion ofimmune cells or presence of specific immune cell populations withintumors following the administration of BO-112 formulation. Depletion ofCD4positive T cells improves the effect of BO-112 formulation,suggesting the clinical potential for combination therapies of BO-112with drugs that target CD4 Tregs (FIGS. 12A and B). Depletion ofCD8positive T cells appears as decreasing the anti-tumor effect ofBO-112 formulation on mouse tumor models confirming the implication ofthe adaptive immune response in the BO-112 anti-tumor mediated effect/.Moreover, the representation of specific cell populations among TumorInfiltrating Lymphocytes is modified by intratumoral injection of BO-112formulation, with the significant increase not only of CD45/CD8positiveT cells, but also of positivity for CD137, OVA and Trp2 tetramers, PD-1,and interferon gamma expression in CD8positive cells, these effectspartially confirmed in CD4positive cells; in the same model specificCD8+ T cells against the endogenous tumor antigen Trp2 are alsoincreased in the tumor draining lymph node (FIGS. 12C and D).

These data confirm that BO-112 formulation promotes the adaptive immuneresponse against the tumor and is able to induce systemic immunity. Thepotent effect of BO-112 to induce tumor cell death and promote T cellinfiltration and activation provides evidence for clinical combinationwith drugs that target complementary mechanisms of tumor progressionsuch as immune suppression (Chen D S and Mellman I, 2013). Activation ofCD4+ and CD8+ T cells in the tumor after BO-112 therapy demonstrate theactivation of IFN signaling pathway. IFNγ plays a pivotal role inanti-tumor systemic and local immunity but also induces PD-L1 expressionin cancer cells, a mechanism described to impair local tumor immunityand drive immunosuppression (Abiko K et al., 2015). BO-112 intratumoraladministration in different mouse tumor models induces PD-L1 expressionin tumor cells, what supports the rational for combination therapy ofBO-112 with PD-1/PD-L1 targeted drugs. Promising combinations includedrugs that target other immune check point inhibitors, in order tounblock the immune suppression induced by the tumor cells and/or thetumor microenvironment such as CTLA4, Tim-3, LAG3 or IDO.

The potent and direct cytotoxic effect of BO-112 formulation on tumorcells is also detected in cells that do not respond to IFNs (havingrelevant clinical implications for patient selection and BO-112treatment. BO-112 formulation appears triggering tumor cell death alsoindependently from the capability of cancer cells to respond tointerferons (FIG. 13). Loss of function mutations in IFN-gamma receptorsignaling or antigen presenting machinery mediate some cases of primaryresistance and acquired resistance to PD-1 blockade therapy (Zaretsky Jet al., 2016). Activation of the RIG1/MDA5 pathway by BO-112 in tumorcells represents an strategy to overcome the IFN-γ resistance and mightresensitize tumors to autologous CD8+ T cells therapies. Earlychromosomal aberrations could predispose to acquired IFNγ resistance andT cell resistance in certain tumor types, therefore screening of patientmetastases for chromosomal aberration and gene mutations prior toimmunotherapy to define the risk of resistance development may be usefulfor the selection of patients that will benefit from BO-112 therapy.

The literature provides several evidences human tissue specimens oncells within tumors (including melanoma, breast invasive carcinoma,prostate adenocarcinoma, lung adenocarcinoma, and colorectaladenocarcinoma) harbour alterations in JAK1 and JAK2, includingloss-of-function alterations in either JAK1 or JAK2 that wouldputatively diminish JAK1 or JAK2 signaling (homodeletions, truncatingmutations, or gene or protein downregulation). These evidences may beuseful for the selection of patients that, in view of their mutationalburden with tumor, would particularly benefit from BO-112 therapy.

Example 3: Clinical Studies Using BO-112 Formulations

The pre-clinical studies involving the administration of BO-112 inanimal models can be used as basis for establishing clinical studies inwhich BO-112 is administered (for example, through intratumoraldelivery) seeking a safer and more focused enhancement of local andsystemic antitumor effects. The potential of BO-112 intratumoraladministration as an immune-modulatory treatment, as well as itstoxicity/safety profile, is being analysed in this first in human, proofof concept, clinical trial (NCT02828098). Patients with cancer lesionswhich are accessible for intratumoral injection, such as malignant solidtumors and palpable cutaneous/sub-cutaneous or lymph node metastases (>1cm) and from whom biopsies can be obtained, are treated with BO-112using intra-tumoral injections at different dose levels (0.6 mg-1 mg ofpoly(I:C) content in each dose, 1-3 doses with one dose/week) startingfrom BO-112 preparations containing poly(I:C) molecules at aconcentration of at least >0.5-mg/mL. The injected lesions are biopsiedbefore treatment is started and at 7-14 days after the last dose, oralternatively after 6 weeks, for example. The patients are evaluated inparallel with respect to signs of clinical relevance and innate oradaptive immune system response and signaling pathways. BO-112pharmacokinetics and circulating cytokines such type I Interferon,TNF-alpha, and IL-6 are also evaluated in plasma.

Preliminary results show that patients did not experience relevanttoxicity with the exception of two episodes of thrombocytopenia, thatwere recovered without treatment. BO-112 was not detectable inbloodstream following intratumoral delivery. BO-112 has determined bothstatistically and therapeutically relevant changes in all patients forat least one of the following tumor-relevant criteria: size of injectedtumor, appearance or not of metastasis, apoptosis and necrosis of cancercells (demonstrating immunogenic cell death triggered in tumors byBO-112 formulations), increase of CD4-positive tumor infiltrating Tcells, increase of CD8-positive tumor infiltrating T cells, increase incirculating immune cells (including NK cells, dendritic cells,monocytes, and/or T regulatory cells, total or specific sub-populationspresenting specific markers such as CD16 or PD-1).

When the BO-11X formulation (or a BO-11Xm formulation) is clinicallyadministered by intratumoral injection, apoptosis and/or necrosis isobserved in the tumor, possibly as a results of the direct antitumoraleffects combined with activation of anti-tumor responses by other cellsresident in the tumor (e.g. by promoting the presentation of tumorantigens to dendritic cells). and cascade may also lead to recruitmentand/or activation of immune cells, in particular CD4+ and CD8+ T cellsinto the tumor mass promoting an immune effect against the tumor,contributing to the cytotoxic effect of BO-11X. Systemic changes inimmune cell populations such as CD8, CD4, CD4 Treg, NK T, NK, CD16+monocytes, or DCs, are also triggered that could be capable of mountingboth primary and memory anti-tumoral responses.

The BO-11X formulations (or a BO-11Xm formulation) can be alsoadministered as a single agent or in combination with other anti-cancertherapies such as checkpoint inhibitors or other immuno-oncology agents(in addition or not to chemotherapy and radiotherapy) given via otherroutes of administration and with different frequency in regimens thatinvolve not only subsequent intra-tumoral injections but also byincluding alternative administration routes such as by sub-cutaneous orintramuscular administration, within one or more cycles of treatments(FIGS. 14A and 14B, also including anti-CTLA4, anti-PD-1 or anti-PD-L1concurrent therapies). A cycle of BO-11X treatment can be established asa pre-defined number of intra-tumoral injections in the same or indifferent tumor lesions (from one up to four or more consecutive onesover one, two, three, four or more weeks).

In a second part of the first-in-human clinical trial referred to above,patients who are not responding to treatment with an anti-PD1 antibodyare given multiple dose fo BO-112 in addton to continued treatment withthe anti-PD1 antibody (see FIG. 14B). Clinical response to treatment, asmeasured by overall tumor burden based on imaging techniques, as well asbiological activity as assessed in tumor tissue and circulating immunecells are evaluated in the trial (see FIG. 15).

that can be followed by a number of intra-muscular or sub-cutaneousinjections (from one up to seven or more consecutive ones over one,four, seven, fifteen or more weeks), repeating such cycle with the sameregimen (or by extending the interval between injections) depending ofpatient responses. The BO-11X formulations in these intra-muscular orsub-cutaneous injections will possibly be diluted before administrationin order to provide lower doses of poly(I:C) molecules, when compared tointra-tumoral injections (e.g. 50%, 25%, 10%, 5%, 1% of the dose that isintra-tumorally injected) but they may still allow sustaining theinitial therapeutic and/or immunomodulating effect of intra-tumoralinjections. In addition to the parameters mentioned above, clinicalresponse may also be evaluated through physical examination, CT-scan,MRI-scan or other imaging techniques for evaluating tumor burden.

Alternatively, the BO-11X formulations (or BO-11Xm formulations) may beadministered ex vivo, using cells that are isolated from a patient, inparticular for improving maturation, efficacy, and/or activation ofspecific immune cells, such as Dendritic Cells (FIG. 14C). This approachmay be applied for in vitro maturation of dendritic cells, as describedin the literature for neoantigen-, adoptive T cell-, cell lysate-,cancer-, gynaecological diseases, and/or infection-related uses(Vanderlocht J et al., 2010; Gallois A and Bhardwaj N, 2013; Da Silva DM et al., 2015; Fritsch E F et al., 2014; Gonzalez F E et al., 2014; TaiL H et al. 2013; Hammerich L et al., 2015; Hervas-Stubbs S et al., 2012;Ochoa M C et al., 2017; Osada T et al., 2015; Perica K et al, 2015).Also in this case, the BO-11X formulations in these in vitro/ex vivomethods will be possibly diluted before administration in order toprovide lower doses of poly(I:C) molecules, when compared tointra-tumoral injections (e.g. 50%, 25%, 10%, 5%, 1% of the dose that isintra-tumorally injected, but possibly within the range of doses andconcentration that are used for validating BO-11X formulation incell-based models, as disclosed in Example 1). These dosages may stillallow providing the therapeutic and/or immunomodulating effect that isappropriate for such therapeutic approaches, with or without laterapplying a cycle of BO-11X treatments in the same patient.

By using any of the routes of administrations and regimen listed above,BO-11X formulations (or BO-11Xm formulations, such as BO-112formulations) can be validated taking into account one or more combinedcriteria that are measured in the patients using biopsies, bloodsamples, and/or other clinical criteria (see above and FIG. 15A). Thus,aside from direct evaluation of tumor size and metastasis, thetherapeutic efficacy of BO-11X formulations can be determined in methodswherein at least one of the following three general criteria areevaluated: direct cytotoxicity on cancer cells (apoptosis and necrosisof cancer cells), increase of tumor infiltrating, immune cells (such asCD4-positive and/or CD8-positive tumor infiltrating T cells), increasein immune cells that circulates in blood (total populations or specificsub-populations of lymphocytes, NK cells, monocytes, dendritic cells,macrophages, B cells, etc.), and/or presenting some differentialexpression pre-versus post-treatment only in either responding ornon-responding patients (as determined by RNA sequencing or other masssequencing approach).

This validation process may also involve follow-up at the molecularlevel by, for example, screening the mRNA and/or protein expression ofspecific sets of proteins such that not only the efficacy of thetreatment can be confirmed but also whether other treatment (previouslyfound ineffective) can be applied since BO-11X (or BO-11Xm)administration has enhanced the patient responsiveness. Examples of suchsignatures and alternative regimes that can be identified in BO-11X orBO-11Xm treated patients, are available in the literature for instancewith respect to Interferon expression and/or other markers to beidentified and acquired resistance to PD-1-based therapies for potentialrescue of patients (Zaretsky J et al., 2016; Masucci G et al., 2016).Thus, safety, efficacy, and immune-biological data will provide therationale for choosing the BO-11X (or BO-11Xm) dosage for futureclinical trials, using BO-11X formulations such as BO-112 alone and/orin combination with other drugs, standard-of-care protocols, orimmunotherapies that can provide further therapeutic benefits (FIG.15B).

The therapeutic efficacy of BO-112 formulation is under clinicalevaluation, with two initial examples that further illustrate therelevant clinical effect (FIGS. 16 and 17). Thesepreliminary resultsconfirms the efficacy of BO-112 in reducing cancer lesions,demonstrating safety and the intended biological effect, with or withoutconcurrent administration of anti-CTLA4, anti-PD1, and/or anti-PD-L1antibodies, with the potential effect to address cancer patientsrefractory to checkpoint therapy and inducing immunogenic cell death andanti-tumor immunity. Moreover, these preliminary results show thatpatients did not experience clinically relevant toxicity with theexception of two cases of thrombocytopenia, both of which recovered (onewith transfusion of platelets the other without treatment). BO-112 wasnot detectable in the bloodstream following intratumoral delivery.BO-112 has demonstrated therapeutically relevant changes in all patientsfor at least one of the following tumor-relevant criteria: size ofinjected tumor, apoptosis and necrosis of cancer cells, increase ofCD4-positive tumor infiltrating T cells, increase of CD8-positive tumorinfiltrating T cells, increase in circulating immune cells (including NKcells, dendritic cells, monocytes, and/or T regulatory cells, total orspecific sub-populations presenting specific markers such as CD16 orPD-1).

Example 4: Evaluation of Biological Effects Due to the Administration ofBO-112 Formulations in Patients

Materials & Methods

The pre- and post-treatment biopsies from the injected metastatic lesionare obtained, in order to analyse apoptosis, necrosis, immune infiltrateand inflammatory gene RNA expression signatures (for example, using theNanostring platform and specific panels of human genes such as thoselisted by the manufacturer as PanCancer Pathways, PanCancer ImmuneProfiling, or PanCancer Progression Panels). Pharmacokinetics, serumcytokines and circulating immune cells are sequentially studied in pre-and post-treatment blood samples.

Using the nSolver Analysis Software (NanoString, Inc.), counts werefirst normalized to the geometric mean of the negative control spikedinto the assay to correct for experimental variability. Then, thegeometric mean was used to compute the normalization factor of thepositive control and, finally, normalized to housekeeping genes builtinto the Human Immunology panel. The detailed normalization analysisguidelines can be found on the NanoString Technologies website[http://www.NanoString.com]. The normalized data were measured ascounts. Normalized data of the nSolver procedure were log-transformed(with base 2) for the 750 endogenous genes.

nCounter chips data for the 12 patients were obtained pre- andpost-treatment. The differential expressions were evaluated using apaired t-test for each gene. The design matrix used was:

$\begin{pmatrix}{pre}_{1} \\{post}_{1} \\{pre}_{2} \\{post}_{2} \\{pre}_{3} \\{post}_{3} \\\ldots \\\ldots \\\ldots \\{pre}_{12} \\{post}_{12}\end{pmatrix} = {\begin{pmatrix}{- 1} & 1 & 0 & 0 & \ldots & 0 \\1 & 1 & 0 & 0 & \ldots & 0 \\{- 1} & 0 & 1 & 0 & \ldots & 0 \\1 & 0 & 1 & 0 & \ldots & 0 \\{- 1} & 0 & 0 & 1 & \ldots & 0 \\1 & 0 & 0 & 1 & \ldots & 0 \\\ldots & \ldots & \ldots & \ldots & \; & \ldots \\{- 1} & 0 & 0 & 0 & \ldots & 1 \\1 & 0 & 0 & 0 & \ldots & 1\end{pmatrix}\begin{pmatrix}\beta_{0} \\\beta_{1} \\\beta_{2} \\\beta_{3} \\\ldots \\\beta_{12}\end{pmatrix}}$

Where β₀ is the differential expression between pre- and post-treatment,and β₁ to β₁₃. Gene modified to higher expression after treatment showedpositive values of fold change. A linear model for nCounter data wasconstructed using the limma package. The p-values were corrected formultiple comparisons using the Benjamini and Hochberg's method. Thegenes with a significance value of p<0.05 were clustered. All geneexpression analyses were performed in R.

Results

Initially, the gene expression studies using cells obtained from BO-112treated patients has allowed identifiing the down- or up-regulation of alimited number of genes, as summarized in table I.

TABLE I Gene Uniprot Gene Description Symbol code (regulation afterBO-112 treatment) CYFIP2 Q96F07 cytoplasmic FMR1 interacting protein 2(down- regulated) LRRN3 Q9H3W5 leucine rich repeat neuronal 3(down-regulated) IL8_2 ???? interleukin 8 (up-regulated) IRAK2 O43187interleukin-1 receptor-associated kinase 2(up- regulated) SLC11A1 P49279Solute carrier family 11 (proton-coupled divalent metal iontransporters), member 1, natural resistance-associated macrophageprotein 1 (up- regulated) CD36 P16671 CD36 molecule (thrombospondinreceptor) (up- regulated) BTK Q06187 Bruton agammaglobulinemia tyrosinekinase (up- regulated) LY96 Q9Y6Y9 Lymphocyte antigen 96 (up-regulated)CD163 Q86VB7 CD163 molecule (up-regulated) CCL7 P80098 Chemokine (C-Cmotif) ligand 7(up-regulated) IFI35 P80217 Interferon-induced protein 35(up-regulated) CXCL1 P09341 Chemokine (C-X-C motif) ligand 1 (melanomagrowth stimulating activity, alpha) (up-regulated) CTSL P07711 cathepsinL1 (up-regulated) TLR2 O60603 toll-like receptor 2, CD282 (up-regulated)CCL3 P10147 chemokine (C-C motif) ligand 3 (up-regulated) IL8 P10145interleukin 8 (up-regulated) LAIR2 Q6ISS4 Leukocyte-associatedimmunoglobulin-like receptor 2, CD306 (up-regulated) OAS3 Q9Y6K52′-5′-oligoadenylate synthetase 3, 100 kDa (up- regulated) IL1R2 P27930interleukin 1 receptor, type II, CD121b (up- regulated) IL10 P22301interleukin 10 (up-regulated) CREB5 Q02930 cAMP responsive elementbinding protein 5 (up- regulated) MARCO Q9UEW3 macrophage receptor withcollagenous structure (up-regulated) MSR1 P21757 macrophage scavengerreceptor 1, CD205 (up- regulated)

The genes are selected present (log 2) fold change, p-value and adjustedp-value. From this analysis, 23 genes were found differentiallyexpressed between pre- and post-treatment (p-value<0.05) within tumors(thus incuding potentially both cancer cells and infiltrating immunecells). The genes listed in the above Table I may associated to one ormore of the GO (Gene Ontology) classes that are associated to themodulation (but preferably activation or increase) of the frequency,rate or extent of an immune response or defense (against a present orpotential internal and/or invasive threat), general immunological and/orinflammatory processes, or other response to a organic, inorganic, orbiological agent. Such GO classes include those having the systematicnames: M14329, M11976, M10574, M13496, M12904, M13657, M12866, M10700,M16728, and M16306. In particular, three genes (CCL7, MARCO, MSR1) arerelated specifically to macrophages, one (OAS3) to activated DC, and one(LAIR2) to Th2 T cells. Two further genes (CD163, CD36) correspond toreceptors involved in phagocytosis.

Alternatively, these genes may be analyzed on the basis of their proteinlocalization and/or enzymatic activity. In a first group of up-regulatedgenes, the corresponding protein is an enzyme whose action can beinhibited by small molecules, these chemical having in many casestherapeutic properties by altering an immune response and/or responseagainst cancer, associated or not to TLR3. For example, IRAK2 is amediator of TLR3 signaling (Jain A et al., 2014), OAS3 is aninterferon-induced dsRNA-activated enzyme which plays a critical role incellular innate response, while cathepsin L1 and BTK are both target ofanticancer and/or immunomodulatory compounds (Feng M et al., 2015;Molina-Cerrillo J et al., 2017; Ping L et al., 2017; Li Y Y et al.,2017; Sudhan D and Siemann D, 2015). In a second group of up-regulatedgenes (MSR1, MARCO, LAIR2, TLR2, CD163, CD36, SLC11A1, LRRN3), thecorresponding protein is a cell surface receptor whose action can beactivated (or inhibited) by various agents (antibodies or smallmolecules), these compounds having, in many cases, therapeuticproperties. In a third group of up-regulated genes (IL10, IL8, CCL3,CCL7, CXCL1, LY96), the corresponding protein is a secreted proteinwhose action or interaction with a cell surface receptor can beinhibited, by various agents (antibodies or small molecules), also thesecompounds having, in many cases, therapeutic properties.

Using these categorization schemes and then testing appropriateantibodies, inhibitors or other commercially available products on celllines, tissue samples or other biological materials, it is possible todefine how BO-11X administration may affect by immune response and/orresponse against cancer by affecting set(s) of genes, associated or notto TLR3-related activities, immune cells, and/or response againstcancer. The approach described above may lead to a series of furtherembodiments of the inventions wherein a BO-11X formulation (including aBO-11Xm formulation) is used in methods for treating a patient sufferingfrom a disease (such as cancer) and/or preventing a disease (such ascancer or an infection) after determining the combined presence (and/orabsence) of expression at the RNA and/or protein level for one or moregenes in cells or tissues of the patient (such as a tumor, a bloodsample, or a blood fraction), post- or pre-treatment with such aformulation. These methods may allow therefore defining a geneexpression signature that is associated to:

-   -   (i) the therapeutically effective amount of a BO-11X formulation        (or a BO-11Xm formulation);    -   (ii) the therapeutically relevant biomarker(s) that predicts        that a subject may have an anti-tumor or anti-infective response        after the treatment with a BO-11X formulation (or a BO-11Xm        formulation):    -   (iii) the therapeutically effective amount of a BO-11X        formulation (or BO-11Xm formulation) that may allow a subject        responding to the treatment with a compound after the treatment        with BO-11X formulation (or BO-11Xm formulation); and/or    -   (iv) The therapeutically relevant biomarker(s) that predicts        that a subject may respond to the treatment with a compound        after the treatment with a BO-11X formulation (or a BO-11Xm        formulation).

Thus, any of the genes of Table I can be used for the uses and methodsas summarized in (i)-(iv) above, using the means for detecting/measuringthe RNA and/or protein expression of any of these genes that arecommercially available and applicable in common technologies such asRT-PCR or antibody-based methods). In particular, the coordinated up-and down regulation of selected (sets of) genes of Table I can be usedfor defining the biological response to BO-11X (and in particularBO-112) when therapeutically administered, and associated to themodulation of TLR3-mediated activities, interferon induction, and/orimmune response, in patients presenting a specific disease, and inparticular specific cancer-related indications, clinical stages, and/orcombination with other drugs. In addition to the products applicable tothe technologies listed above (such as nucleic acid probes orantibodies), the biological activity of such genes is often welldefined, with a variety of commercially and technically agents that areavailable for testing appropriately the potential interaction with theadministration of a BO-11X formulation, such as BO-112.

Moreover, further therapeutic agents that are available for targetingproteins whose expression is affected by the treatment with a BO-11Xformulation (or BO-11Xm formulation). Drugs targeting any of theaforementioned genes might be advantageously used in combination withBO-11X formulations by administering them either within the samecomposition or in distinct compositions.

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The invention claimed is:
 1. An aqueous composition comprising one ormore particles wherein: (i) each particle comprises a complex of atleast one double-stranded polyribonucleotide, or a salt or solvatethereof, and at least one water-soluble, linear polyethyleneimine, or asalt and/or solvate thereof, wherein (a) the double-strandedpolyribonucleotide is polyinosinic-polycytidylic acid [poly(I:C)], or asalt or solvate thereof, wherein at least 40% of the poly(I:C) have atleast 850 base pairs, and at least 50% of poly(I:C) have between 400 and5000 base pairs; and between 5% and 60% of poly(I:C) have less than 400base pairs; between 15% and 30% of poly(I:C) have between 400 and 850base pairs, between 10% and 70% of poly(I:C) have between 850 and 5000base pairs; between 0% and 10% of poly(I:C) have more than 5000 basepairs, and (b) the average molecular weight of the water-soluble, linearpolyethyleneimine is between 17 and 23 kDa and has a polydispersityindex of <1.5; (ii) at least 90% of the particles have a mono-modaldiameter distribution of below 300 nm; (iii) the one or more particleseach have a z-average diameter of 80+/−20 nm measured according to ISOstandard 22412:2017, and each particle diameter has a polydispersityindex between 0.1 and 0.6; (iv) the polyinosinic-polycytidylic acid[poly(I:C)] concentration is at least 0.5 mg/mL; (v) the composition hasa pH of between 3.0+/−0.2 and an osmolality of between 300 and 310mOsm/kg; and (vi) the composition has a zeta potential between 35 mV and50 mV measured according to ISO standard 13099-2:2012.
 2. The aqueouscomposition according to claim 1, wherein said particles have a mediandiameter (D50%) of 85+/−20 nm.
 3. The aqueous composition according toclaim 1, wherein the polydispersity index of each particle diameter isbetween 0.2 and 0.3.
 4. The aqueous composition according to claim 1,further comprising at least one pharmaceutically acceptable carrier,organic solvent, excipient and/or adjuvant.
 5. The aqueous compositionaccording to claim 1, further comprising glucose or mannitol at aconcentration of between 1 and 10% (weight/volume).
 6. The aqueouscomposition according to claim 1, wherein the composition has a pH ofbetween 3.0+/−0.2 and an osmolality of between 300 and 310 mOsm/kg.
 7. Acomposition obtainable by lyophilisation of the aqueous compositionaccording to claim
 1. 8. A method for treating cancer, comprisingadministering to a subject in need thereof, an effective amount of anaqueous composition according to claim
 1. 9. The method according toclaim 8, wherein the aqueous composition is an injectable composition,optionally comprising a pharmaceutically acceptable carrier, excipientand/or adjuvant.
 10. The method according to claim 8, wherein theaqueous composition is administered by injection comprising intratumoralinjection, peritumoral injection, injection into the skin or injectioninto an internal organ or tissue.
 11. The method according to claim 8,wherein the aqueous composition is administered in combination with asecond therapeutic agent.
 12. The method according to claim 11, whereinthe second therapeutic agent is selected from an immune checkpointmolecule targeting agent, CAR-T cells, cancer antigen vaccines, oragents that target regulatory T cells, metabolic enzymes, or DNA repairand/or DNA replication.
 13. The method according to claim 12, whereinthe immune checkpoint molecule targeting agent is selected fromanti-CTLA4, anti-PD1, and anti-PDL1.
 14. The method according to claim8, wherein said aqueous composition is administered to a subjectaccording to an administration regimen comprising: (i) at least a firstintratumoral injection into a first lesion; and (ii) at least a secondintratumoral injection into one or more additional lesions.
 15. Themethod according to claim 14, wherein the aqueous composition isadministered prior to or subsequent to administering a secondtherapeutic agent, wherein the second therapeutic agent is administeredinto the same and/or the one or more additional lesion by sub-cutaneous,intratumoral, peritumoral or intramuscular injection.
 16. The methodaccording to claim 15, wherein said second therapeutic agent isadministered to a subject after determining the subject is resistant,insensitive, or non-responsive to said second therapeutic agent.
 17. Themethod according to claim 15, wherein said second therapeutic agent isadministered to a subject after determining if, following theadministration of a composition according to claim 1, there is anincrease in number of circulating immune cells.
 18. The method accordingto claim 15, wherein said at least second intratumoral, peritumoral,sub-cutaneous or intra-muscular injection is performed at least 24 hoursafter the at least first intratumoral injection.
 19. The methodaccording to claim 8, wherein the cancer is a solid tumor.
 20. Themethod according to claim 19, wherein the solid tumor is a carcinoma,glioma, melanoma, or sarcoma.